US20090215988A1 - Soluble, Degradable Poly(Ethylene Glycol) Derivatives for Controllable Release of Bound Molecules into Solution - Google Patents

Soluble, Degradable Poly(Ethylene Glycol) Derivatives for Controllable Release of Bound Molecules into Solution Download PDF

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US20090215988A1
US20090215988A1 US12/414,621 US41462109A US2009215988A1 US 20090215988 A1 US20090215988 A1 US 20090215988A1 US 41462109 A US41462109 A US 41462109A US 2009215988 A1 US2009215988 A1 US 2009215988A1
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J. Milton Harris
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Nektar Therapeutics
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    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08GMACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32Polymers modified by chemical after-treatment
    • C08G65/329Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33396Polymers modified by chemical after-treatment with organic compounds containing nitrogen having oxygen in addition to nitrogen
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08GMACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32Polymers modified by chemical after-treatment
    • C08G65/329Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33303Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group
    • C08G65/33317Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group heterocyclic

Definitions

  • This invention relates to active derivatives of poly(ethylene glycol) and related hydrophilic polymers having a reactive moiety at one end of the polymer chain suitable for chemical coupling to another molecule.
  • PEG poly(ethylene glycol)(PEG), which is also known as poly(ethylene oxide) (PEO), to molecules and surfaces is of great utility in biotechnology.
  • PEG poly(ethylene glycol)
  • PEO poly(ethylene oxide)
  • This polymer can be represented in brief form as “HO-PEG-OH” where it is understood that the -PEG- symbol represents the following structural unit:
  • n typically ranges from approximately 10 to approximately 2000.
  • PEG is commonly used as methoxy-PEG-OH, or “mPEG”, in which one terminus is the relatively inert methoxy group, while the other terminus is a hydroxyl group that is subject to ready chemical modification.
  • PEG is also commonly used in branched forms that can be prepared by addition of ethylene oxide to various polyols, such as glycerol, pentaerythritol and sorbitol.
  • polyols such as glycerol, pentaerythritol and sorbitol.
  • pentaerythritol a polyol prepared from pentaerythritol is shown below:
  • Such branched polyethylene glycols can be represented in general form as R(-PEG-OH) z in which R represents the central “core” molecule, such as glycerol or pentaerythritol, and z represents the number of arms extending therefrom.
  • PEG is a well-known polymer having the following properties: solubility in water and in many organic solvents, lack of toxicity, and lack of immunogenicity.
  • One use of PEG is to covalently attach the polymer to insoluble molecules to make the resulting PEG-molecule conjugate soluble. For example, Greenwald, Pendri and Bolikal in J. Org. Chem., 60, 331-336 (1995), have shown that the water-insoluble drug, taxol, when coupled to PEG, becomes water-soluble.
  • an activated derivative of PEG is the succinimidyl “active ester” of carboxymethylated PEG as disclosed by K. Iwasaki and Y. Iwashita in U.S. Pat. No. 4,670,417. This chemistry can be illustrated with the active ester reacting with amino groups of a protein (the succinimidyl group is represented as “NHS” and the amino-containing protein is represented as PRO-NH 2 ):
  • Attachment of a PEG derivative to a substance can have a somewhat unpredictable impact on the substance. Proteins, small drugs, and the like may have reduced biological activity when conjugated to a PEG derivative, although in some cases, activity may be increased.
  • succinimidyl succinate active ester “mPEG-SS” (the succinimidyl group is represented as NHS):
  • the mPEG-SS active ester is useful for coupling because it reacts rapidly with amino groups on proteins and other molecules to form an amide linkage (—CO—NH—).
  • a problem has been reported with the mPEG-SS active ester, as noted in U.S. Pat. No. 4,670,417. Since this compound possesses an ester linkage in the backbone that remains after coupling to an amine group, such as in a protein (represented as PRO-NH 2 ):
  • the present invention provides chemically active polyethylene glycols and related polymers that are suitable for coupling to other molecules.
  • the polyethylene glycol derivatives provided herein when covalently attached to another molecule, form water-soluble conjugates in which the linkage between the polymer and the bound molecule is subject to predetermined cleavage. This allows for controlled delivery of the bound molecule into its surrounding environment.
  • the polymers of the invention contain weak, hydrolytically unstable linkages near the reactive end of the polymer that provide for a sufficient circulation period for a drug-PEG conjugate, followed by hydrolytic breakdown of the conjugate and release of the bound molecule.
  • also forming part of the present invention are (i) methods of preparing the degradable active PEGs described herein and related polymers, (ii) conjugates, and (iii) methods for preparing such PEG conjugates.
  • the PEG and related polymer derivatives of the invention are capable of imparting improved water solubility, increased size, a slower rate of kidney clearance, and reduced immunogenicity to a conjugate formed by covalent attachment thereto, while also providing for controllable hydrolytic release of the bound molecule into an aqueous environment—by virtue of the design of the linkages provided herein.
  • the invention can be used to enhance the solubility and blood circulation lifetime of drugs in the bloodstream, while also delivering a drug into the bloodstream that, subsequent to hydrolysis, is substantially free of PEG.
  • the invention is especially useful in those cases where drugs, when permanently conjugated to PEG, demonstrate reduced activity.
  • a degradable PEG as provided herein, such drugs can maintain their therapeutic activity when in conjugated form, by virtue of the cleavable nature of the PEGs of the invention.
  • POLY is a linear or branched polyethylene glycol of molecular weight from 300 to 100,000 daltons. POLY can also be a related non-peptidic polymer as described in greater detail below;
  • y is the number of chemically active groups on POLY and is the number of molecules that can be bound to POLY;
  • W is a hydrolytically unstable group
  • T is a reactive group (i.e., is reactive with Y);
  • Y—P′ represents a molecule for conjugation to POLY, where Y is a reactive group that is reactive with T, and P′ is the portion of the molecule that is to be bound and released, including, for example, a peptide P′ in which Y is an amine moiety and T is a PEG activating moiety reactive with an amine;
  • X is the new linkage formed by reaction of Y and T;
  • G and I are new groups formed by hydrolysis of W.
  • hydrolytically unstable groups include carboxylate esters, phosphate esters, acetals, imines, orthoesters, peptides and oligonucleotides.
  • the variables, T and Y are groups that react with each other. There are many examples of such groups known in organic chemistry. Some examples include the following combinations: active esters reacting with amines, isocyanates reacting with alcohols and amines, aldehydes reacting with amines, epoxides reacting with amines, and sulfonate esters reacting with amines, among others.
  • P′ examples include peptides, oligonucleotides and other pharmaceuticals.
  • Examples of X, the linkage resulting from reaction of Y and T, include amide from reaction of active esters with amine, urethane from reaction of isocyanate with hydroxyl, and urea from reaction of amine with isocyanate.
  • Examples of G and I the new groups formed upon hydrolysis of W, are alcohol and acid from hydrolysis of carboxylate esters, aldehyde and alcohol from hydrolysis of acetals, aldehyde and amine from hydrolysis of imines, phosphate and alcohol from hydrolysis of phosphate esters, amine and acid from hydrolysis of peptide, and phosphate and alcohol from hydrolysis of oligonucleotides.
  • the released exemplary peptide contains no polymer, in this case, mPEG. Rather, the released peptide contains an additional short molecular fragment, which is sometimes called a “tag”.
  • This tag is the portion of the linkage opposite the PEG from the hydrolytically unstable linkage. In the above example, the tag portion is the ‘HO—(CH 2 ) 3 —O—C(O)—’ which remains attached to the released peptide.
  • the invention provides activated PEGs and other related polymers containing hydrolytically unstable linkages.
  • the polymers are useful, when conjugated to a drug or other molecule, for controlled delivery of such molecule to its surrounding environment.
  • linkages including ester linkages, are suitable for use as the hydrolytically unstable linkage as provided herein.
  • ester linkages of the present polymers in contrast to mPEG-SS and mPEG-SG, provide for variation and control of the rate of hydrolytic degradation.
  • FIG. 1 is a MALDI-MS spectrum of the molecular weight distribution of an mPEG-HBA-subtilisin conjugate at 1 day after preparation as described in Example 8.
  • FIG. 2 is a MALDI-MS spectrum of the molecular weight distribution of an mPEG-HBA-subtilisin conjugate at 8 days after preparation as described in Example 8.
  • FIG. 3 is a MALDI-MS spectrum of the molecular weight distribution of an mPEG-HBA-subtilisin conjugate at 14 days after preparation as described in Example 8.
  • PEG poly(ethylene glycol).
  • other related polymers are also suitable for use in the practice of the invention in place of PEG and that the use of the term PEG or poly(ethylene glycol) is intended to be inclusive and not exclusive in this respect.
  • Poly(ethylene glycol) is preferred in the practice of the invention.
  • PEG is used in biological applications because it has properties that are highly desirable and is generally approved for biological or biotechnological applications.
  • PEG typically is clear, colorless, odorless, soluble in water, stable to heat, inert to many chemical agents, does not hydrolyze or deteriorate, and is non-toxic.
  • Poly(ethylene glycol) is considered to be biocompatible, which is to say that PEG is capable of coexistence with living tissues or organisms without causing harm. More specifically, PEG is not immunogenic, which is to say that PEG does not tend to produce an immune response in the body.
  • the PEG When attached to a moiety having some desirable function in the body, the PEG tends to mask the moiety and can reduce or eliminate an immune response so that an organism can tolerate the presence of the moiety, in PEGylated form. Accordingly, the activated PEGs of the invention should be substantially non-toxic and not tend to substantially produce an immune response or cause clotting or other undesirable effects.
  • Water-soluble polymers other than PEG are suitable for similar modification in accordance with the invention described herein.
  • These other polymers include poly(vinyl alcohol) (“PVA”); other poly(alkylene oxides) such as poly(propylene glycol) (“PPG”) and the like; and poly(oxyethylated polyols) such as poly(oxyethylated glycerol), poly(oxyethylated sorbitol), and poly(oxyethylated glucose), and the like.
  • PVA poly(vinyl alcohol)
  • PPG poly(propylene glycol)
  • poly(oxyethylated polyols) such as poly(oxyethylated glycerol), poly(oxyethylated sorbitol), and poly(oxyethylated glucose), and the like.
  • the polymers can be homopolymers or random or block copolymers and terpolymers based on monomers of the above polymers, straight chain or branched, substituted or unsubstituted, e.g., similar to mPEG and other capped, mono-functional PEGs having a single active site available for attachment to a linker.
  • PVP and poly(oxazoline) are well-known polymers in the art and their preparation and use in the syntheses described herein for embodiments based upon mPEG should be readily apparent to the skilled artisan.
  • PAcM and its synthesis and use are described in U.S. Pat. Nos. 5,629,384 and 5,631,322, the contents of which are incorporated herein by reference in their entireties.
  • drug any substance intended for the diagnosis, cure, mitigation, treatment, or prevention of disease in humans and other animals, or used to otherwise enhance physical or mental well-being.
  • the invention is useful, for example, for delivery of biologically active substances that generally have some activity or function in a living organism or in a substance taken from a living organism.
  • group is all somewhat synonymous in the chemical arts and are used in the art and herein to refer to distinct, definable portions or units of a molecule and to units that perform some function or activity and are reactive with other molecules or portions of molecules.
  • linkages are used herein to refer to groups that are normally formed as the result of a chemical reaction. Herein, such linkages are typically covalent linkages.
  • “Hydrolytically stable linkage” means a linkage that is stable in water and does not react with water under useful or normal conditions of pH for an extended period of time, potentially indefinitely.
  • “Hydrolytically unstable linkage” is a linkage or functionality that reacts with water, typically causing a molecule to separate (or cleave) into two or more components. Such a linkage is said to be “subject to hydrolysis” and to be “hydrolysable”. The time it takes for the linkage to react with water is referred to as the rate of hydrolysis and is usually measured in terms of its half-life.
  • the invention includes poly(ethylene glycols) containing, for example, an ester group as the weak linkage and a succinimidyl ester as the reactive group useful for coupling to amine-containing molecules.
  • the resulting conjugates can be delivered in vivo or into a substance taken from a living entity.
  • Exemplary polymers, and in particular, linkages, are provided below.
  • the invention also encompasses poly(ethylene glycols) containing an ester group as the weak linkage and an isocyanate as the reactive group useful for coupling to amine- and alcohol-containing molecules, as exemplified below.
  • the invention also includes poly(ethylene glycols) containing an acetal as the weak linkage and a succinimidyl ester as the reactive group useful, for example, for coupling to an amine-containing molecule.
  • the invention encompasses poly(ethylene glycols) containing an imine group as the weak linkage and a succinimidyl ester as the reactive group, e.g., useful for coupling to, for example, an amine-containing molecule.
  • the invention also includes poly(ethylene glycols) containing a phosphate ester group as the weak linkage and a succinimidyl ester as the reactive group, e.g., useful for coupling to an amine-containing molecule.
  • the invention includes poly(ethylene glycols) containing an ester-linked amino acid as the weak linkage and a succinimidyl ester as the reactive group, e.g., useful for coupling to an amine-containing molecule.
  • poly(ethylene glycols) containing an ester-linked amino acid as the weak linkage and a succinimidyl ester as the reactive group, e.g., useful for coupling to an amine-containing molecule.
  • the invention includes poly(ethylene glycols) containing a peptide as the weak linkage and a succinimidyl ester as the reactive group useful for coupling to an amine-containing molecule.
  • the invention includes poly(ethylene glycols) containing an oligonucleotide forming the weak linkage and a succinimidyl ester as the reactive group, e.g., useful for coupling to an amine-containing molecule.
  • poly(ethylene glycols) containing an oligonucleotide forming the weak linkage and a succinimidyl ester as the reactive group, e.g., useful for coupling to an amine-containing molecule.
  • polymers for use in the invention can be straight chain or branched. That is to say, branched activated PEGs can be prepared in accordance with the invention where such PEGs possess weak linkages near the reactive end of the polymer for controlled hydrolytic degradation.
  • Illustrative branched PEGs are described in International Publication No. WO 96/21469, entitled, “Multi-Armed, Monofunctional, and Hydrolytically Stable Derivatives of Poly(Ethylene Glycol) and Related Polymers For Modification of Surfaces and Molecules”, filed Jan. 11, 1996, the content of which is incorporated herein by reference in its entirety. Branched PEGs such as these can then be modified in accordance with the present teachings.
  • the invention is illustrated with respect to several particular examples below, including determination of hydrolysis half-lives for representative hydrolyzable polymer derivatives and conjugates.
  • CH 3 O-PEG 3000 -O—(CH 2 ) 1,2 —COOH (3.0 g, 1 mmol, mPEG-CM or mPEG-PA) was azeotropically dried with 60 ml of toluene under N 2 . After two hours, the solution was cooled to room temperature, followed by injection of a solution of thionyl chloride (2 ml, 4 mmol) in CH 2 Cl 2 . The resulting solution was stirred at room temperature overnight. The solvent was condensed on a rotary evaporator and the residual syrup was dried in vacuo for about four hours over P 2 O 5 powder.
  • Glycolic acid (0.2 g, 2.7 mmole) was azeotropically distilled with 70 ml of 1,4-dioxane and the distillation was stopped when approximately 20 ml of solution remained. The solution was slowly cooled to room temperature under N 2 . The glycolic acid/dioxane solution was then added to the dried PEG acyl chloride. After the PEG was dissolved, 0.6 ml of dry triethylamine was injected to the system (precipitate formed immediately) and the solution was stirred overnight. The salt was removed by filtration and the filtrate was condensed on a rotary evaporator at 55° C. and dried in vacuo.
  • the crude product was then dissolved in 100 ml of distilled water and the pH of the solution was adjusted to 3.0.
  • the aqueous phase was extracted three times with a total of 80 ml of methylene chloride.
  • the combined organic phase was dried over sodium sulfate, filtered to remove salt, condensed on a rotary evaporator, and added to 100 ml of ethyl ether. The precipitate was collected by filtration and dried in vacuo.
  • Difunctional carboxymethyl PEG 20,000 -ester benzyl glycolate Difunctional carboxymethyl PEG 20,000 (4 gram, 0.4 mmol acid group), benzyl glycolate (0.6 mmol), dimethylaminopyridine (0.44 mmol), 1-hydroxybenzotriazole (0.4 mmol) and dicyclohexylcarbodiimide (0.56 mmol) were dissolved in 40 ml of methylene chloride. The solution was stirred at room temperature under N 2 overnight. The solvent was then removed under vacuum and the resulting residue was added to 20 ml of toluene at 40° C. The undissolved solid was removed by filtration and the filtrate was added to 200 ml of ethyl ether. The precipitate was collected by filtration and dried in vacuo.
  • Difunctional carboxymethyl PEG-ester benzyl glycolate 20,000 (3 gram) and Pd/C (10%, 0.8 gram) were added to 30 ml of 1,4-dioxane. The mixture was shaken with H 2 (40 psi) at room temperature overnight. The Pd/C was removed by filtration and the solvent was condensed by rotary evaporation. The resulting syrup was added to 100 ml of ether. The precipitated product was collected by filtration and dried in vacuo.
  • CH 3 O-PEG-O—(CH 2 ) n —COOH 3000 (3.0 g, 1 mmol) was azeotropically dried with 60 ml of toluene under N 2 . After two hours, the solution was slowly cooled to room temperature. To the resulting solution was added thionyl chloride solution (3 ml, 6 mmol) in CH 2 Cl 2 , and the solution was stirred overnight. The solvent was condensed by rotary evaporation and the syrup was dried in vacuo for about four hours over P 2 O 5 powder.
  • 3-hydroxybutyric acid (HBA, 0.30 g, 2.7 mmol) was azeotropically dried with 70 ml of 1,4-dioxane on a rotary evaporator. The distillation was stopped when approximately 20 ml of solution remained. This solution was then slowly cooled to room temperature under N 2 , and the solution was added to the dried PEG acyl chloride. After the PEG was dissolved, 0.6 ml of dry triethylamine was injected to the system (precipitate formed immediately) and the solution was stirred overnight. The salt was removed by filtration and the filtrate was condensed on a rotary evaporator at 55° C. and dried in vacuo.
  • the crude product was then dissolved in 100 ml of distilled water and the pH of the solution was adjusted to 3.0.
  • the aqueous phase was extracted three times with a total of 80 ml of methylene chloride.
  • the organic phase was dried over sodium sulfate, filtered to remove salt, condensed on a rotary evaporator, and added to 100 ml of ethyl ether.
  • the precipitate was collected by filtration and dried in vacuo. Yield 2.76 g (92%).
  • CM-HBA-NHS or PA-HBA-NHS mPEG 3000 -ester butyric acid (1 g, approx., 0.33 mmol, CM-HBA-COOH or PA-HBA-COOH) and 42 mg N-hydroxysuccinimide (NHS) (0.35 mmol) were dissolved in 30 ml of dry methylene chloride. To this solution was added dicyclohexylcarbodiimide (DCC) (80 mg, 0.38 mmole) in 5 ml of dry methylene chloride. The solution was stirred under nitrogen overnight and the solvent removed by rotary evaporation.
  • DCC dicyclohexylcarbodiimide
  • the residual syrup was re-dissolved in 10 ml of dry toluene and the insoluble solid was removed by filtration. The solution was then precipitated into 100 ml of dry ethyl ether. The precipitate was collected by filtration and dried in vacuo.
  • Determination of hydrolysis half-lives of PEG active esters Measurements were conducted using a HP8452a UV-VIS spectrophotometer. In an experiment, 1 mg of a given PEG active ester was dissolved in 3.0 ml of buffer solution and shaken promptly to obtain dissolution as soon as possible. The solution was then transferred into a UV cuvette and the absorbance at 260 nm (for NHS ester) or at 402 nm (for the p-nitrophenyl ester) was followed as a function of time. The hydrolytic half-life was determined from the first order kinetic plot (natural logarithm of final absorbance minus absorbance at the time t versus time).
  • subtilisin with an illustrative PEG derivative: To a subtilisin solution (1 ml, 2 mg/ml in 0.2M boric buffer, pH 8.0) was added 15 mg mPEG 3000 -CM-HBA-NHS. The solution was placed in an automatic shaker at room temperature. At predetermined time periods, 50 ⁇ l of the solution was removed and preserved in a refrigerator for MALDI-TOF MS measurement.
  • TOF PerSeptive Biosystems' Voyager linear time-of-flight
  • Protein samples were dissolved in deionized H 2 O or 50 mM NaCl solution to a concentration of approximately 10 pmol/ ⁇ l.
  • the matrix 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), was dissolved in an 80:20 by volume ratio of acetonitrile to deionized water at a concentration of 10 mg/ml. 1 ⁇ l of the solution was deposited on the sample plate and then mixed with 1 ⁇ l of matrix solution. The sample was allowed to crystallize by solvent evaporation under ambient conditions.
  • FIGS. 1 through 3 MALDI-MS spectra of the molecular weight distribution of mPEG-HBA and its subtilisin conjugate are shown in FIGS. 1 through 3 .
  • FIG. 1 is at 1 day.
  • FIG. 2 is at 8 days.
  • FIG. 3 is at 14 days.
  • mPEG-CM-SSP and 20% PEG 20,000 (wt) were dissolved in 10 mM phosphate buffer (pH 7.2) and a series of ampoules were sealed with each containing about 0.25 ml of above solution.
  • the ampoules were stored as two groups, with one group at room temperature and the other at 37° C. For each measurement, one ampoule in each group was opened and the solution was analyzed.
  • the concentrations of mPEG-CM-SSP and its hydrolysis product were determined by HPLC-GPC (Ultrahydrogel 250 column, Waters; 5 mM phosphate buffer pH 7.2 as mobile phase). The hydrolytic half-life was obtained from the slope of the natural logarithm of C at the time t minus C at infinite time versus time, assuming 1st order kinetics.

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Abstract

PEG and related polymer derivatives having weak, hydrolytically unstable linkages near the reactive end of the polymer are provided for conjugation to drugs, including proteins, enzymes, small molecules, and others. These derivatives provide a sufficient circulation period for a drug-PEG conjugate, followed by hydrolytic breakdown of the conjugate and release of the bound molecule. In some cases, drugs that demonstrate reduced activity when permanently coupled to PEG maintain a therapeutically suitable activity when coupled to a degradable PEG in accordance with the invention. The PEG derivatives of the invention can be used to impart improved water solubility, increased size, a slower rate of kidney clearance, and reduced immunogenicity to a conjugate formed by attachment thereto. Controlled hydrolytic release of the bound molecule into an aqueous environment can then enhance the drug's delivery profile by providing a delivery system which employs such polymers and utilizes the teachings provided herein.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • This application is a continuation of U.S. patent application Ser. No. 11/051,728, filed Feb. 4, 2005, which is a continuation of U.S. patent application Ser. No. 10/318,322, filed Dec. 12, 2002, now U.S. Pat. No. 6,864,350, which is a continuation of U.S. patent application Ser. No. 09/824,297, filed Apr. 2, 2001, now U.S. Pat. No. 6,515,100, which is a divisional of U.S. patent application Ser. No. 08/937,846, filed Sep. 25, 1997, now U.S. Pat. No. 6,214,966, which claims the benefit of priority to Provisional Patent Application Ser. No. 60/026,716, filed Sep. 26, 1996, the contents of which are all incorporated herein by reference in their entireties.
  • FIELD OF THE INVENTION
  • This invention relates to active derivatives of poly(ethylene glycol) and related hydrophilic polymers having a reactive moiety at one end of the polymer chain suitable for chemical coupling to another molecule.
  • BACKGROUND OF THE INVENTION
  • Chemical attachment of the hydrophilic polymer poly(ethylene glycol)(PEG), which is also known as poly(ethylene oxide) (PEO), to molecules and surfaces is of great utility in biotechnology. In its most common form PEG is a linear polymer terminated at each end with a hydroxyl group:

  • HO—CH2CH2O—(CH2CH2O)n—CH2CH2—OH
  • This polymer can be represented in brief form as “HO-PEG-OH” where it is understood that the -PEG- symbol represents the following structural unit:

  • —CH2CH2O—(CH2CH2O)n—CH2CH2—,
  • where n typically ranges from approximately 10 to approximately 2000.
  • PEG is commonly used as methoxy-PEG-OH, or “mPEG”, in which one terminus is the relatively inert methoxy group, while the other terminus is a hydroxyl group that is subject to ready chemical modification.

  • CH3O—(CH2CH2O)n—CH2CH2—OH mPEG
  • PEG is also commonly used in branched forms that can be prepared by addition of ethylene oxide to various polyols, such as glycerol, pentaerythritol and sorbitol. For example, the four-arm, branched PEG prepared from pentaerythritol is shown below:

  • C(CH2—OH)4 +nC2H4O→C[CH2O—(CH2CH2O)n—CH2CH2—OH]4
  • Such branched polyethylene glycols can be represented in general form as R(-PEG-OH)z in which R represents the central “core” molecule, such as glycerol or pentaerythritol, and z represents the number of arms extending therefrom.
  • PEG is a well-known polymer having the following properties: solubility in water and in many organic solvents, lack of toxicity, and lack of immunogenicity. One use of PEG is to covalently attach the polymer to insoluble molecules to make the resulting PEG-molecule conjugate soluble. For example, Greenwald, Pendri and Bolikal in J. Org. Chem., 60, 331-336 (1995), have shown that the water-insoluble drug, taxol, when coupled to PEG, becomes water-soluble.
  • In related work, Davis et al., in U.S. Pat. No. 4,179,337, have shown that proteins coupled to PEG have enhanced blood-circulation lifetimes due to reduced rate of kidney clearance and reduced immunogenicity. Hydrophobic proteins have been described that, upon coupling to PEG, have increased solubility in water. These and other uses for PEG are described in J. M. Harris, Ed., “Biomedical and Biotechnical Applications of Polyethylene Glycol Chemistry,” Plenum, New York, 1992).
  • To couple PEG to a molecule such as a protein or onto a surface, it is necessary to use an “activated derivative” of the PEG having a functional group at a terminus suitable for reacting with some group on the protein or on the surface (such as an amino group). Among the many useful activated derivatives of PEG is the succinimidyl “active ester” of carboxymethylated PEG as disclosed by K. Iwasaki and Y. Iwashita in U.S. Pat. No. 4,670,417. This chemistry can be illustrated with the active ester reacting with amino groups of a protein (the succinimidyl group is represented as “NHS” and the amino-containing protein is represented as PRO-NH2):

  • PEG-O—CH2—CO2—NHS+PRO-NH2→PEG-O—CH2—CO2—NH-PRO
  • Certain problems have arisen in the art. Some of the functional groups that have been used to activate PEG can result in toxic or otherwise undesirable residues when used for in vivo drug delivery. Some of the linkages that have been devised to attach functional groups to PEG can result in an undesirable immune response. Additionally, certain functional groups do not have appropriate selectivity for reacting with particular groups on proteins and can deactivate the proteins when in conjugated form.
  • Attachment of a PEG derivative to a substance can have a somewhat unpredictable impact on the substance. Proteins, small drugs, and the like may have reduced biological activity when conjugated to a PEG derivative, although in some cases, activity may be increased.
  • Another example of a problem that has arisen in the art is exemplified by the succinimidyl succinate active ester, “mPEG-SS” (the succinimidyl group is represented as NHS):
  • Figure US20090215988A1-20090827-C00001
  • The mPEG-SS active ester is useful for coupling because it reacts rapidly with amino groups on proteins and other molecules to form an amide linkage (—CO—NH—). A problem has been reported with the mPEG-SS active ester, as noted in U.S. Pat. No. 4,670,417. Since this compound possesses an ester linkage in the backbone that remains after coupling to an amine group, such as in a protein (represented as PRO-NH2):

  • mPEG-SS+PRO-NH2→mPEG-OC(O)—CH2CH2—CONH-PRO,
  • the remaining ester linkage is subject to rapid hydrolysis and detachment of PEG from the modified protein. Too rapid a hydrolysis rate can preclude use of a PEG derivative for many applications. Several approaches have been adopted to solve the problem of hydrolytic instability. For example, mPEG succinimidyl carbonate has been proposed, which contains only ether linkages in the polymer backbone and reacts with proteins to form a conjugate that is not subject to hydrolysis.
  • In view of the above, it would be desirable to provide alternative PEG derivatives that are suitable for drug delivery systems, including delivery of proteins, enzymes, and small molecules, or for other biotechnology uses. It would also be desirable to provide alternative PEG derivatives that could enhance drug delivery systems or biotechnology products.
  • SUMMARY OF THE INVENTION
  • In one aspect, the present invention provides chemically active polyethylene glycols and related polymers that are suitable for coupling to other molecules. The polyethylene glycol derivatives provided herein, when covalently attached to another molecule, form water-soluble conjugates in which the linkage between the polymer and the bound molecule is subject to predetermined cleavage. This allows for controlled delivery of the bound molecule into its surrounding environment.
  • The polymers of the invention contain weak, hydrolytically unstable linkages near the reactive end of the polymer that provide for a sufficient circulation period for a drug-PEG conjugate, followed by hydrolytic breakdown of the conjugate and release of the bound molecule.
  • According to yet another aspect, also forming part of the present invention are (i) methods of preparing the degradable active PEGs described herein and related polymers, (ii) conjugates, and (iii) methods for preparing such PEG conjugates.
  • The PEG and related polymer derivatives of the invention are capable of imparting improved water solubility, increased size, a slower rate of kidney clearance, and reduced immunogenicity to a conjugate formed by covalent attachment thereto, while also providing for controllable hydrolytic release of the bound molecule into an aqueous environment—by virtue of the design of the linkages provided herein. The invention can be used to enhance the solubility and blood circulation lifetime of drugs in the bloodstream, while also delivering a drug into the bloodstream that, subsequent to hydrolysis, is substantially free of PEG. The invention is especially useful in those cases where drugs, when permanently conjugated to PEG, demonstrate reduced activity. By using a degradable PEG as provided herein, such drugs can maintain their therapeutic activity when in conjugated form, by virtue of the cleavable nature of the PEGs of the invention.
  • In general form, the polymer derivatives of the invention and their conjugates can be described by the following equations:

  • POLY(-W-T)y+Y—P′→POLY(-W—X—P′)y+H2O→

  • →POLY-Gy+(I—X—P′)
  • In the above equations,
  • “POLY” is a linear or branched polyethylene glycol of molecular weight from 300 to 100,000 daltons. POLY can also be a related non-peptidic polymer as described in greater detail below;
  • “y” is the number of chemically active groups on POLY and is the number of molecules that can be bound to POLY;
  • W is a hydrolytically unstable group;
  • T is a reactive group (i.e., is reactive with Y);
  • Y—P′ represents a molecule for conjugation to POLY, where Y is a reactive group that is reactive with T, and P′ is the portion of the molecule that is to be bound and released, including, for example, a peptide P′ in which Y is an amine moiety and T is a PEG activating moiety reactive with an amine;
  • X is the new linkage formed by reaction of Y and T; and
  • G and I are new groups formed by hydrolysis of W.
  • Examples of hydrolytically unstable groups, W, include carboxylate esters, phosphate esters, acetals, imines, orthoesters, peptides and oligonucleotides. The variables, T and Y, are groups that react with each other. There are many examples of such groups known in organic chemistry. Some examples include the following combinations: active esters reacting with amines, isocyanates reacting with alcohols and amines, aldehydes reacting with amines, epoxides reacting with amines, and sulfonate esters reacting with amines, among others.
  • Examples of P′ include peptides, oligonucleotides and other pharmaceuticals.
  • Examples of X, the linkage resulting from reaction of Y and T, include amide from reaction of active esters with amine, urethane from reaction of isocyanate with hydroxyl, and urea from reaction of amine with isocyanate.
  • Examples of G and I, the new groups formed upon hydrolysis of W, are alcohol and acid from hydrolysis of carboxylate esters, aldehyde and alcohol from hydrolysis of acetals, aldehyde and amine from hydrolysis of imines, phosphate and alcohol from hydrolysis of phosphate esters, amine and acid from hydrolysis of peptide, and phosphate and alcohol from hydrolysis of oligonucleotides.
  • An embodiment of the invention is shown in the following equation demonstrating conjugation of a hydrolyzable methoxy-PEG (mPEG) polymer derivative with a peptide drug, followed by hydrolytic release of the peptide. In the embodiment below, the weak linkage, W, contains a hydrolyzable ester group.
  • Figure US20090215988A1-20090827-C00002
  • The released exemplary peptide contains no polymer, in this case, mPEG. Rather, the released peptide contains an additional short molecular fragment, which is sometimes called a “tag”. This tag is the portion of the linkage opposite the PEG from the hydrolytically unstable linkage. In the above example, the tag portion is the ‘HO—(CH2)3—O—C(O)—’ which remains attached to the released peptide.
  • Thus, the invention provides activated PEGs and other related polymers containing hydrolytically unstable linkages. The polymers are useful, when conjugated to a drug or other molecule, for controlled delivery of such molecule to its surrounding environment. Several types of linkages, including ester linkages, are suitable for use as the hydrolytically unstable linkage as provided herein. However, the ester linkages of the present polymers, in contrast to mPEG-SS and mPEG-SG, provide for variation and control of the rate of hydrolytic degradation.
  • The foregoing and other objects, advantages, and features of the invention, and the manner in which the same are accomplished, will be more readily apparent upon consideration of the following detailed description of the invention taken in conjunction with the accompanying drawings, which illustrate an exemplary embodiment of the invention.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is a MALDI-MS spectrum of the molecular weight distribution of an mPEG-HBA-subtilisin conjugate at 1 day after preparation as described in Example 8.
  • FIG. 2 is a MALDI-MS spectrum of the molecular weight distribution of an mPEG-HBA-subtilisin conjugate at 8 days after preparation as described in Example 8.
  • FIG. 3 is a MALDI-MS spectrum of the molecular weight distribution of an mPEG-HBA-subtilisin conjugate at 14 days after preparation as described in Example 8.
  • DETAILED DESCRIPTION
  • The following detailed description describes various embodiments of the invention as described by the following general equations presented in the preceding summary:

  • Poly(-W-T)y+Y—P′→Poly(-W—X—P′)y+H2O→

  • Poly-Gy+I—X—P′
  • The Polymer
  • In the discussion below, POLY will often be referred to for convenience as PEG or as poly(ethylene glycol). However, it is to be understood that other related polymers are also suitable for use in the practice of the invention in place of PEG and that the use of the term PEG or poly(ethylene glycol) is intended to be inclusive and not exclusive in this respect.
  • Poly(ethylene glycol) is preferred in the practice of the invention. PEG is used in biological applications because it has properties that are highly desirable and is generally approved for biological or biotechnological applications. PEG typically is clear, colorless, odorless, soluble in water, stable to heat, inert to many chemical agents, does not hydrolyze or deteriorate, and is non-toxic. Poly(ethylene glycol) is considered to be biocompatible, which is to say that PEG is capable of coexistence with living tissues or organisms without causing harm. More specifically, PEG is not immunogenic, which is to say that PEG does not tend to produce an immune response in the body. When attached to a moiety having some desirable function in the body, the PEG tends to mask the moiety and can reduce or eliminate an immune response so that an organism can tolerate the presence of the moiety, in PEGylated form. Accordingly, the activated PEGs of the invention should be substantially non-toxic and not tend to substantially produce an immune response or cause clotting or other undesirable effects.
  • Water-soluble polymers other than PEG are suitable for similar modification in accordance with the invention described herein. These other polymers include poly(vinyl alcohol) (“PVA”); other poly(alkylene oxides) such as poly(propylene glycol) (“PPG”) and the like; and poly(oxyethylated polyols) such as poly(oxyethylated glycerol), poly(oxyethylated sorbitol), and poly(oxyethylated glucose), and the like. The polymers can be homopolymers or random or block copolymers and terpolymers based on monomers of the above polymers, straight chain or branched, substituted or unsubstituted, e.g., similar to mPEG and other capped, mono-functional PEGs having a single active site available for attachment to a linker.
  • Specific examples of such suitable polymers include poly(oxazoline), poly(acryloylmorpholine) (“PAcM”), and poly(vinylpyrrolidone)(“PVP”). PVP and poly(oxazoline) are well-known polymers in the art and their preparation and use in the syntheses described herein for embodiments based upon mPEG should be readily apparent to the skilled artisan. PAcM and its synthesis and use are described in U.S. Pat. Nos. 5,629,384 and 5,631,322, the contents of which are incorporated herein by reference in their entireties.
  • DEFINITIONS
  • It should be understood that by “drug” is meant any substance intended for the diagnosis, cure, mitigation, treatment, or prevention of disease in humans and other animals, or used to otherwise enhance physical or mental well-being. The invention is useful, for example, for delivery of biologically active substances that generally have some activity or function in a living organism or in a substance taken from a living organism.
  • The terms “group,” “functional group,” “moiety,” “active moiety,” “reactive site,” and “radical” are all somewhat synonymous in the chemical arts and are used in the art and herein to refer to distinct, definable portions or units of a molecule and to units that perform some function or activity and are reactive with other molecules or portions of molecules.
  • The term “linkage” is used herein to refer to groups that are normally formed as the result of a chemical reaction. Herein, such linkages are typically covalent linkages.
  • “Hydrolytically stable linkage” means a linkage that is stable in water and does not react with water under useful or normal conditions of pH for an extended period of time, potentially indefinitely.
  • “Hydrolytically unstable linkage” is a linkage or functionality that reacts with water, typically causing a molecule to separate (or cleave) into two or more components. Such a linkage is said to be “subject to hydrolysis” and to be “hydrolysable”. The time it takes for the linkage to react with water is referred to as the rate of hydrolysis and is usually measured in terms of its half-life.
  • The Hydrolytically Unstable Linkage (W) and Reactive Groups, T and Y
  • In a particular embodiment, the invention includes poly(ethylene glycols) containing, for example, an ester group as the weak linkage and a succinimidyl ester as the reactive group useful for coupling to amine-containing molecules. The resulting conjugates can be delivered in vivo or into a substance taken from a living entity. Exemplary polymers, and in particular, linkages, are provided below.

  • PEG-W—CO2—NHS
  • where
      • W=—O2C—(CH2)b—O— b=1-5
        • —O—(CH2)bCO2—(CH2)c— b=1-5, c=2-5
        • —O—(CH2)b—CO2—(CH2)c—O— b=1-5, c=2-5
  • The invention also encompasses poly(ethylene glycols) containing an ester group as the weak linkage and an isocyanate as the reactive group useful for coupling to amine- and alcohol-containing molecules, as exemplified below.

  • PEG-W—N═C═O
  • where W=—O—(CH2)b—CO2—(CH2)c— b=1 to 5, c=2 to 5
  • As a further example, the invention also includes poly(ethylene glycols) containing an acetal as the weak linkage and a succinimidyl ester as the reactive group useful, for example, for coupling to an amine-containing molecule.

  • PEG-W—CO2—NHS,
  • where, for example, W equals:
  • Figure US20090215988A1-20090827-C00003
  • In the above illustrative structures,
    o=1-10,
    Z=—O—C6H4— and —O—(CH2)d—CH2— d=1-5
    R′=alkyl or H.
  • In yet another embodiment, the invention encompasses poly(ethylene glycols) containing an imine group as the weak linkage and a succinimidyl ester as the reactive group, e.g., useful for coupling to, for example, an amine-containing molecule.

  • PEG-W—CO2—NHS
  • where W=-Z-CH═N—(CH2)b—O— b=1-5
      • Z=—O—C6H4
        • —O—(CH2)b—CH2— b=1-5
  • According to yet another embodiment, the invention also includes poly(ethylene glycols) containing a phosphate ester group as the weak linkage and a succinimidyl ester as the reactive group, e.g., useful for coupling to an amine-containing molecule.

  • PEG-W—CO2—NHS
  • where W=—(CH2)b—OPO3—(CH2)b′— b and b′=1-5
  • According to yet another exemplary embodiment, the invention includes poly(ethylene glycols) containing an ester-linked amino acid as the weak linkage and a succinimidyl ester as the reactive group, e.g., useful for coupling to an amine-containing molecule. An advantage of this derivative is that its hydrolytic breakdown results in a biologically acceptable amino acid attached to the released molecule:

  • PEG-W—CO2—NHS
  • where W=—O—(CH2)b—CO2—(CH2)b′—CH(NH-t-Boc)- b=1-5,
      • b′=1-5, and
      • t-Boc=(CH3)3C—O—CO—.
  • In yet a further embodiment, the invention includes poly(ethylene glycols) containing a peptide as the weak linkage and a succinimidyl ester as the reactive group useful for coupling to an amine-containing molecule. An advantage of this particular type of derivative is that its hydrolytic breakdown typically results in a biologically acceptable peptide fragment attached to the released molecule:

  • PEG-W—CO2—NHS
  • where W=—C(O)(NH—CHR—CO)a—NH—CHR— a=2-20
      • R=the set of substituents typically found on α-amino acids
  • In yet another exemplary embodiment, the invention includes poly(ethylene glycols) containing an oligonucleotide forming the weak linkage and a succinimidyl ester as the reactive group, e.g., useful for coupling to an amine-containing molecule. An advantage of this particular type of derivative is that its hydrolytic breakdown results in a biologically acceptable oligonucleotide fragment attached to the released molecule:

  • PEG-W—CO2—NHS
  • where W=oligonucleotide.
  • As previously described, polymers for use in the invention can be straight chain or branched. That is to say, branched activated PEGs can be prepared in accordance with the invention where such PEGs possess weak linkages near the reactive end of the polymer for controlled hydrolytic degradation. Illustrative branched PEGs are described in International Publication No. WO 96/21469, entitled, “Multi-Armed, Monofunctional, and Hydrolytically Stable Derivatives of Poly(Ethylene Glycol) and Related Polymers For Modification of Surfaces and Molecules”, filed Jan. 11, 1996, the content of which is incorporated herein by reference in its entirety. Branched PEGs such as these can then be modified in accordance with the present teachings.
  • The invention is illustrated with respect to several particular examples below, including determination of hydrolysis half-lives for representative hydrolyzable polymer derivatives and conjugates.
  • EXAMPLES Example 1
  • Figure US20090215988A1-20090827-C00004
  • CH3O-PEG3000-O—(CH2)1,2—COOH (3.0 g, 1 mmol, mPEG-CM or mPEG-PA) was azeotropically dried with 60 ml of toluene under N2. After two hours, the solution was cooled to room temperature, followed by injection of a solution of thionyl chloride (2 ml, 4 mmol) in CH2Cl2. The resulting solution was stirred at room temperature overnight. The solvent was condensed on a rotary evaporator and the residual syrup was dried in vacuo for about four hours over P2O5 powder.
  • Glycolic acid (0.2 g, 2.7 mmole) was azeotropically distilled with 70 ml of 1,4-dioxane and the distillation was stopped when approximately 20 ml of solution remained. The solution was slowly cooled to room temperature under N2. The glycolic acid/dioxane solution was then added to the dried PEG acyl chloride. After the PEG was dissolved, 0.6 ml of dry triethylamine was injected to the system (precipitate formed immediately) and the solution was stirred overnight. The salt was removed by filtration and the filtrate was condensed on a rotary evaporator at 55° C. and dried in vacuo. The crude product was then dissolved in 100 ml of distilled water and the pH of the solution was adjusted to 3.0. The aqueous phase was extracted three times with a total of 80 ml of methylene chloride. The combined organic phase was dried over sodium sulfate, filtered to remove salt, condensed on a rotary evaporator, and added to 100 ml of ethyl ether. The precipitate was collected by filtration and dried in vacuo.
  • Yield 2.55 g (85%). 1H NMR (DMSO-d6): δ 3.5 (br m, PEG), 4.3-4.6 (s, PEGOCH2COOCH 2COOH), 2.59 (t, PEGOCH2CH 2COO(PA)), 4.19 (s, PEGOCH 2COO(CM)).
  • Example 2
  • Figure US20090215988A1-20090827-C00005
  • Difunctional carboxymethyl PEG20,000-ester benzyl glycolate: Difunctional carboxymethyl PEG 20,000 (4 gram, 0.4 mmol acid group), benzyl glycolate (0.6 mmol), dimethylaminopyridine (0.44 mmol), 1-hydroxybenzotriazole (0.4 mmol) and dicyclohexylcarbodiimide (0.56 mmol) were dissolved in 40 ml of methylene chloride. The solution was stirred at room temperature under N2 overnight. The solvent was then removed under vacuum and the resulting residue was added to 20 ml of toluene at 40° C. The undissolved solid was removed by filtration and the filtrate was added to 200 ml of ethyl ether. The precipitate was collected by filtration and dried in vacuo.
  • Yield: 4 gram (100%). 1H NMR (DMSO-d6): δ 3.5 (br m, PEG), 4.81 (s, PEGOCH 2COOCH2COOCH2C6H5), 5.18 (s, PEGOCH2COOCH2COOCH 2C6H5), 7.37 (s, PEGOCH2COOCH2COOCH2C6H5), 4.24 (s, PEGOCH2COOCH2COOCH2C6H5).
  • Difunctional carboxymethyl PEG-ester benzyl glycolate 20,000 (3 gram) and Pd/C (10%, 0.8 gram) were added to 30 ml of 1,4-dioxane. The mixture was shaken with H2 (40 psi) at room temperature overnight. The Pd/C was removed by filtration and the solvent was condensed by rotary evaporation. The resulting syrup was added to 100 ml of ether. The precipitated product was collected by filtration and dried in vacuo.
  • Yield 2.4 gram (80%). 1H NMR (DMSO-d6): δ 3.5 (br m, PEG), 4.56 (s, PEGOCH 2COOCH 2COOH), 4.20 (s, PEGOCH 2COOCH2COOH).
  • Example 3
  • Figure US20090215988A1-20090827-C00006
  • CH3O-PEG-O—(CH2)1,2—COO—CH2—COOH (1 g, approx. 0.33 mmol) and 42 mg N-hydroxysuccinimide (NHS) (0.35 mmol) was dissolved in 30 ml of dry methylene chloride. To this was added dicyclohexylcarbodiimide (DCC) (80 mg, 0.38 mmol) in 5 ml of dry methylene chloride. The solution was stirred under nitrogen overnight and the solvent was removed by rotary evaporation. The resulting syrup was re-dissolved in 10 ml of dry toluene and the insoluble solid was filtered off. The solution was then precipitated into 100 ml of dry ethyl ether. The precipitate was collected by filtration and dried in vacuo.
  • Yield 0.95 g (95%). 1H NMR (DMSO-d6): δ 3.5 (br m, PEG), 5.15-5.21 (s, PEGOCH2COOCH 2COONHS), 2.67 (t, PEGOCH2CH 2COO(PA)), 4.27 (s, PEGOCH 2COO ppm(CM)), 2.82 (s, NHS, 4H).
  • Example 4
  • Figure US20090215988A1-20090827-C00007
  • CH3O-PEG-O—(CH2)1,2—COO—CH2—COOH (1.5 g, approx. 0.5 mmol), 140 mg p-nitrophenol (1 mmol) and 65 mg dimethylaminopyridine (DMAP) (0.525 mmol) were dissolved in 30 ml of dry methylene chloride. To the resulting solution was added dicyclohexylcarbodiimide (DCC) (120 mg, 0.575 mmole) in 5 ml of dry methylene chloride. The solution was stirred under nitrogen overnight and the solvent was removed by rotary evaporation. The resulting syrup was redissolved in 10 ml of dry toluene and the insoluble solid was removed by filtration. Then the solution was precipitated into 100 ml of dry ethyl ether. The product was reprecipitated with ethyl ether, then collected by filtration and dried in vacuo.
  • Yield 1.425 g (95%). 1H NMR (DMSO-d6): δ 3.5 (br m, PEG), 5.01 (s, PEGOCH2COOCH 2COONP), 2.69 (t, PEGOCH 2CH2COO(PA)), 8.35 & 7.48 (d&d, Ha & Hb in NP, 4H).
  • Example 5
  • Figure US20090215988A1-20090827-C00008
  • CH3O-PEG-O—(CH2)n—COOH 3000 (3.0 g, 1 mmol) was azeotropically dried with 60 ml of toluene under N2. After two hours, the solution was slowly cooled to room temperature. To the resulting solution was added thionyl chloride solution (3 ml, 6 mmol) in CH2Cl2, and the solution was stirred overnight. The solvent was condensed by rotary evaporation and the syrup was dried in vacuo for about four hours over P2O5 powder. 3-hydroxybutyric acid (HBA, 0.30 g, 2.7 mmol) was azeotropically dried with 70 ml of 1,4-dioxane on a rotary evaporator. The distillation was stopped when approximately 20 ml of solution remained. This solution was then slowly cooled to room temperature under N2, and the solution was added to the dried PEG acyl chloride. After the PEG was dissolved, 0.6 ml of dry triethylamine was injected to the system (precipitate formed immediately) and the solution was stirred overnight. The salt was removed by filtration and the filtrate was condensed on a rotary evaporator at 55° C. and dried in vacuo. The crude product was then dissolved in 100 ml of distilled water and the pH of the solution was adjusted to 3.0. The aqueous phase was extracted three times with a total of 80 ml of methylene chloride. The organic phase was dried over sodium sulfate, filtered to remove salt, condensed on a rotary evaporator, and added to 100 ml of ethyl ether. The precipitate was collected by filtration and dried in vacuo. Yield 2.76 g (92%). 1H NMR (DMSO-d6): δ 3.5 (br m, PEG), 2.54 (d, PEGOCH2COOCH(CH3)CH 2COOH), 5.1 (h, PEGOCH2COOCH(CH3)CH2COOH), 1.2 (d, PEG-OCH2COOCH(CH 3)CH2COOH), 2.54 (t, PEGOCH2CH 2COO(PA)), 4.055 (s, PEGOCH 2COO(CM)).
  • mPEG-ester butyric acid NHS ester (CM-HBA-NHS or PA-HBA-NHS): mPEG3000-ester butyric acid (1 g, approx., 0.33 mmol, CM-HBA-COOH or PA-HBA-COOH) and 42 mg N-hydroxysuccinimide (NHS) (0.35 mmol) were dissolved in 30 ml of dry methylene chloride. To this solution was added dicyclohexylcarbodiimide (DCC) (80 mg, 0.38 mmole) in 5 ml of dry methylene chloride. The solution was stirred under nitrogen overnight and the solvent removed by rotary evaporation. The residual syrup was re-dissolved in 10 ml of dry toluene and the insoluble solid was removed by filtration. The solution was then precipitated into 100 ml of dry ethyl ether. The precipitate was collected by filtration and dried in vacuo.
  • Yield 0.94 g (94%). 1H NMR (DMSO-d6): δ 3.5 (br m, PEG), 3.0-3.2 (m, COOCH(CH3)CH 2COONHS), 5.26 (h, COOCH(CH3)CH2—COONHS), 1.3 (d, COOCH(CH 3)CH2COONHS), 2.54 (t, OCH2CH 2COO(PA)), 4.1 (s, OCH 2COO(CM)), 2.81 (s, NHS).
  • Example 6 Determination of the Hydrolytic Half-Lives of the Ester Linkages Contained in Four Exemplary PEG-PEG Conjugates
  • Figure US20090215988A1-20090827-C00009
  • A. Preparation of CH3O-PEG-O—(CH2)n—COO—CH2—CONH-PEG-OCH3 (PEG-PEG conjugates): CH3O-PEG3000-O—(CH2)n—COO—CH2—COOH (0.5 g), 1 equiv. of mPEG2000-NH2 and 1 equiv. of 1-hydroxybenzotriazole (HOBT) were dissolved in 50 ml of methylene chloride. To this solution was added one equivalent of dicyclohexylcarbodiimide (DCC) and the solution was stirred at room temperature overnight. The solvent was partially evaporated, the insoluble salt was filtered, and the filtrate was added into a large excess of ethyl ether. The precipitate was collected by filtration and dried in vacuo. Yield: 0.8 g (95%). 1H MNR (DMSO-d6): δ 3.5 (br m, PEG), 2.34 (t, —CONHCH 2CH2O-PEG-).
  • B. Determination of hydrolytic half-lives of PEG ester conjugates formed by reaction of CM-GA, PA-GA, CM-HBA or PA-HBA with a PEG amine: The conjugates from A. above and 20 wt % PEG 20,000 (as an internal standard) were dissolved in a buffer solution. The concentrations of each of the conjugates (C) and their hydrolysis products were monitored by HPLC-GPC (Ultrahydrogel 250 column, 7.8×300 mm, Waters) at predetermined times. The hydrolytic half-lives were obtained from the slope of the natural logarithm of C at the time, t, minus C at infinite time versus time assuming 1st order kinetics.
  • TABLE 1
    HYDROLYSIS HALF-LIVES
    (DAYS, UNLESS NOTED OTHERWISE) OF THE ESTER
    LINKAGES CONTAINED IN THE ABOVE CONJUGATES. (±10%)
    Double-Ester PEG Used to Form Conjugate
    CM-GA PA-GA CM-HBA PA-HBA
    pH 7.0 7.0 8.1 7.0 8.1 7.0 8.1
    23° C. 3.2 43 6.5 15 120
    37° C. 14 h 7.6 14 112
    50° C.  4 h 2.2 5 58
  • Example 7 Determination of Hydrolysis Half-Lives of the Ester Linkages Contained in Four Exemplary PEG Reagents
  • Figure US20090215988A1-20090827-C00010
  • Determination of hydrolysis half-lives of PEG active esters: Measurements were conducted using a HP8452a UV-VIS spectrophotometer. In an experiment, 1 mg of a given PEG active ester was dissolved in 3.0 ml of buffer solution and shaken promptly to obtain dissolution as soon as possible. The solution was then transferred into a UV cuvette and the absorbance at 260 nm (for NHS ester) or at 402 nm (for the p-nitrophenyl ester) was followed as a function of time. The hydrolytic half-life was determined from the first order kinetic plot (natural logarithm of final absorbance minus absorbance at the time t versus time).
  • TABLE 2
    HYDROLYSIS HALF-LIVES OF SUCCINIMIDYL ACTIVE ESTERS
    (R = NHS) AND P-NITROPHENYL ACTIVE ESTERS (R = NP)
    OF PEG-ESTER ACIDS AT PH 8.1 AND ROOM TEMPERATURE
    R CM-GA-R PA-GA-R CM-HBA-R PA-HBA-R
    NHS 11 s 11 s 12 min 12 min
    NP  7 min  7 min
  • Example 8 Hydrolytic Release of PEG from a PEG-Protein Conjugate by MALDI-TOF Mass Spectrometry
  • Modification of subtilisin with an illustrative PEG derivative: To a subtilisin solution (1 ml, 2 mg/ml in 0.2M boric buffer, pH 8.0) was added 15 mg mPEG3000-CM-HBA-NHS. The solution was placed in an automatic shaker at room temperature. At predetermined time periods, 50 μl of the solution was removed and preserved in a refrigerator for MALDI-TOF MS measurement.
  • MALDI Analyses: MALDI spectra were measured on a PerSeptive Biosystems' Voyager linear time-of-flight (TOF) instrument. Briefly, a nitrogen laser lamda=337 nm, 10 ns pulse width) was used to generate ions which were extracted with a potential of 30 kV. Ions drifted through a 1.3 m drift tube and were monitored in positive ion mode.
  • Protein samples were dissolved in deionized H2O or 50 mM NaCl solution to a concentration of approximately 10 pmol/μl. The matrix, 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), was dissolved in an 80:20 by volume ratio of acetonitrile to deionized water at a concentration of 10 mg/ml. 1 μl of the solution was deposited on the sample plate and then mixed with 1 μl of matrix solution. The sample was allowed to crystallize by solvent evaporation under ambient conditions.
  • MALDI-MS spectra of the molecular weight distribution of mPEG-HBA and its subtilisin conjugate are shown in FIGS. 1 through 3. Each of the figures represents the solution at a different time subsequent to preparation. FIG. 1 is at 1 day. FIG. 2 is at 8 days. FIG. 3 is at 14 days.
  • Example 9
  • Figure US20090215988A1-20090827-C00011
  • CH3O-PEG-O—CH2—COOH 5000 (3.0 g, 0.6 mmol), 2-(2-pyridyldithio)ethanol (342 mg, 1.5 mmol), DMAP (180 mg, 1.44 mmol) and HOBT (93 mg, 0.61 mmol) were dissolved in 60 ml of dichloromethane. To this solution was added DCC (138 mg, 0.66 mmol) in 5 ml of dichloromethane. The solution was stirred at room temperature under N2 overnight. The solvent was removed by rotary evaporation and 15 ml of toluene was added to the residue. After all PEG was dissolved, the solution was filtered to remove dicyclohexyl urea. To the solution was added 45 ml of methylene chloride and the solution was washed with sodium acetate buffer (0.1M, pH 5.0) containing 10% sodium chloride. The organic phase was dried over anhydrous sodium sulfate, filtered to remove salt, condensed on a rotary evaporator, and precipitated into 100 ml of ethyl ether. The product was collected by filtration and dried in vacuo. Yield 2.85 g (95%). 1H NMR (DMSO-d6): δ 3.5 (br m, PEG), 4.11 (s, PEGOCH 2COO—), 4.30 (t, COOCH 2CH2SS—) 7.29 (t, one aromatic proton), 7.77 (t+d, two aromatic protons), 8.46 (d, one aromatic proton).
  • Example 10 Determination of the Hydrolysis Half-Life of an Ester Linkage in an Illustrative PEG Derivative
  • Figure US20090215988A1-20090827-C00012
  • mPEG-CM-SSP and 20% PEG 20,000 (wt) (as internal standard) were dissolved in 10 mM phosphate buffer (pH 7.2) and a series of ampoules were sealed with each containing about 0.25 ml of above solution. The ampoules were stored as two groups, with one group at room temperature and the other at 37° C. For each measurement, one ampoule in each group was opened and the solution was analyzed. The concentrations of mPEG-CM-SSP and its hydrolysis product were determined by HPLC-GPC (Ultrahydrogel 250 column, Waters; 5 mM phosphate buffer pH 7.2 as mobile phase). The hydrolytic half-life was obtained from the slope of the natural logarithm of C at the time t minus C at infinite time versus time, assuming 1st order kinetics.
  • TABLE 3
    HYDROLYTIC HALF-LIVES (DAYS) OF
    THE ESTER IN MPEG-CM-SSP. (10%)
    pH 5.5 pH 7.0
    Room temperature 107 18
    37° C. 20 2.9
  • Example 11
  • Figure US20090215988A1-20090827-C00013
  • (a) Preparation of CH3O-PEG-OCH2CH2CO2-PEG-OBz
  • In a 100 ml round-bottom flask, a solution of CH3O-PEG-O—(CH2)n—CO2H (MW=2000, 2 g, 1 mmol) was dissolved in toluene and azeotropically dried for two hours. After slowly cooling to room temperature, the solution was added to thionyl chloride (3 ml, 6 mmole) in methylene chloride and then stirred under N2 overnight. The solvent was then removed by rotary evaporation and the residual syrup was dried in vacuo for about four hours over P2O5 powder. To the solid was added 5 ml of anhydrous methylene chloride and a solution (20 ml) of azeotropically dried BzO-PEG-OH (MW=3400, 2.04 g, 0.60 mmol) in toluene. To the resulting solution was added 0.6 ml of freshly distilled triethylamine and the solution was stirred overnight. The triethylamine salt was removed by filtration and the crude product was precipitated with ethyl ether and collected by filtration. The mixture was then purified by ion-exchange chromatography (DEAE sepharose fast flow column, Pharmacia). Pure CH3O-PEG-O—(CH2)n—CO2-PEG-OBz was obtained. Yield: 2.6 g (80%). NMR (DMSO-d6): δ 3.5 (br m, PEG), 2.55 (t, —OCH2CH 2COOPEG-), 4.14 (s, -PEGOCH 2COOPEG-), 4.13 (t, -PEGOCH2CH2—COOCH 2CH2OPEG-), 4.18 (t, -PEGOCH2—COOCH 2CH2OPEG), 4.49 (s, -PEG-O—CH 2—C6H5), 7.33 (s+com, -PEG-O—CH2—C6 H 5).
  • (b) Preparation of CH3O-PEG-O—(CH2)n—CO2-PEG-OH
  • A solution of 2 g of CH3O-PEG-O—(CH2)n—CO2-PEG-OBz in 1,4-dioxane was hydrogenolyzed with H2 (2 atm) on 1 gram Pd/C (10%) overnight. The catalyst was removed by filtration, the solvent was condensed under vacuum and the solution was added to ethyl ether. The product was collected by filtration and dried under vacuum at room temperature to yield: 1.5 g (75%) of CH3O-PEG-O—(CH2)n—CO2-PEG-OH. NMR (DMSO-d6): δ 3.5 (br m, PEG), 2.55 (t, —OCH2CH 2COOPEG-), 4.14 (s, -PEG-OCH 2COOPEG-), 4.13 (t, -PEGOCH2CH2COOCH 2CH2OPEG-), 4.18 (t, -PEGOCH2—COOCH 2CH2OPEG).
  • (c) Preparation of CH3O-PEG-O—(CH2)n—CO2-PEG-OCOONHS
  • CH3O-PEG-O—(CH2)n—CO2-PEG-OH 5400 (1.25 g, 0.23 mmole) was azeotropically distilled with 100 ml acetronitrile and then cooled to room temperature. To this mixture was added disuccinimidyl carbonate (245 milligram, 0.92 mmole) and 0.1 ml of pyridine, and the solution was stirred at room temperature overnight. The solvent was then removed under vacuum, and the resulting solid was dissolved in 35 ml of dry methylene chloride. The insoluble solid was removed by filtration, and the filtrate was washed with pH 4.5 sodium chloride saturated acetate buffer. The organic phase was dried over anhydrous sodium sulfate, filtered, condensed by rotary evaporation, and precipitated into ethyl ether. The product was collected by filtration and dried in vacuo.
  • Yield: 1.20 g (96%), 100% substitution of succimidyl carbonate and no reagent left. NMR (DMSO-d6): δ 3.5 (br m, PEG), 2.55 (t, —OCH2CH 2COOPEG-), 4.14 (s, -PEG-OCH 2COOPEG-), 4.13 (t, -PEGOCH2CH2COOCH2CH2OPEG-), 4.18 (t, -PEGOCH2—COOCH 2CH2OPEG), 4.45 (t, -PEGO-CH2CH 2OCONHS), 2.81 (s, NHS).
  • The invention has been described in particular exemplified embodiments. However, the foregoing description is not intended to limit the invention to the exemplified embodiments, and the skilled artisan should recognize that variations can be made within the scope and spirit of the invention as described in the foregoing specification. On the contrary, the invention includes all alternatives, modifications, and equivalents that may be included within the true spirit and scope of the invention as defined by the appended claims.

Claims (12)

1. A polymer having the following structure:

POLY(-W-T)n
wherein:
POLY is a four-arm, branched PEG having a molecular weight from 300 to 100,000 daltons;
W is a linkage comprising an ester;
T is a succinimidyl ester; and
n is four.
2. The polymer of claim 1, wherein the four-arm, branched PEG has a pentaerythritol core.
3. The polymer of claim 1, wherein the linkage comprising an ester is —O—(CH2)n′—CO2—(CH2)m—, wherein n′ is 1 to 5 and m is 2 to 5.
4. The polymer of claim 1, wherein the succinimidyl ester is —CO2-succinimidyl.
5. A method of forming a coupled product, the method comprising reacting a polymer having the following structure: POLY(-W-T)n, with an amine-containing molecule under conditions effective to couple the polymer to the amine-containing molecule to thereby form the coupled product,
wherein:
POLY is a branched PEG having a molecular weight from 300 to 100,000 daltons;
W is a linkage comprising an ester;
T is a succinimidyl ester; and
n is the number of chemically reactive groups on the polymer.
6. The method of claim 5, wherein the branched PEG is a four-arm, branched PEG and n is four.
7. The method of claim 6, wherein the four-arm, branched PEG has a pentaerythritol core.
8. The method of claim 5, wherein the linkage comprising an ester is —O—(CH2)n′—CO2—(CH2)m—, wherein n′ is 1 to 5 and m is 2 to 5.
9. The method of claim 5, wherein the succinimidyl ester is —CO2-succinimidyl.
10. The method of claim 5, wherein the amine-containing molecule is a peptide.
11. The method of claim 5, wherein: the branched PEG is a four-arm, branched PEG having a pentaerythritol core; n is four; the succinimidyl ester is —CO2-succinimidyl; and the amine-containing molecule is a peptide.
12. A composition comprising the coupled product formed by the method of claim 5.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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Families Citing this family (288)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100361933B1 (en) * 1993-09-08 2003-02-14 라 졸라 파마슈티칼 컴파니 Chemically defined nonpolymeric bonds form the platform molecule and its conjugate
US6057287A (en) 1994-01-11 2000-05-02 Dyax Corp. Kallikrein-binding "Kunitz domain" proteins and analogues thereof
US20020064546A1 (en) * 1996-09-13 2002-05-30 J. Milton Harris Degradable poly(ethylene glycol) hydrogels with controlled half-life and precursors therefor
WO1998012274A1 (en) * 1996-09-23 1998-03-26 Chandrashekar Pathak Methods and devices for preparing protein concentrates
US8003705B2 (en) * 1996-09-23 2011-08-23 Incept Llc Biocompatible hydrogels made with small molecule precursors
US6258351B1 (en) * 1996-11-06 2001-07-10 Shearwater Corporation Delivery of poly(ethylene glycol)-modified molecules from degradable hydrogels
US20050158273A1 (en) * 1997-09-25 2005-07-21 Harris J. M. Soluble, degradable polyethylene glycol) derivatives for controllable release of bound molecules into solution
US6514534B1 (en) 1998-08-14 2003-02-04 Incept Llc Methods for forming regional tissue adherent barriers and drug delivery systems
US6632457B1 (en) * 1998-08-14 2003-10-14 Incept Llc Composite hydrogel drug delivery systems
EP1137373A4 (en) * 1998-12-04 2004-05-19 Chandrashekhar P Pathak Biocompatible crosslinked polymers
US6458953B1 (en) * 1998-12-09 2002-10-01 La Jolla Pharmaceutical Company Valency platform molecules comprising carbamate linkages
CN1762990A (en) * 1999-06-08 2006-04-26 拉卓拉药物公司 Valency platform molecules comprising aminooxy groups
AU2001273387A1 (en) * 2000-07-12 2002-01-21 Gryphon Therapeutics, Inc. Polymer-modified bioactive synthetic chemokines, and methods for their manufacture and use
US7118737B2 (en) 2000-09-08 2006-10-10 Amylin Pharmaceuticals, Inc. Polymer-modified synthetic proteins
CN1454097A (en) * 2000-09-08 2003-11-05 格莱风治疗公司 Polymer-modified synthetic proteins
US7265186B2 (en) * 2001-01-19 2007-09-04 Nektar Therapeutics Al, Corporation Multi-arm block copolymers as drug delivery vehicles
TWI246524B (en) * 2001-01-19 2006-01-01 Shearwater Corp Multi-arm block copolymers as drug delivery vehicles
IL142875A (en) * 2001-04-30 2009-08-03 Avigdor Shafferman Cholinesterases conjugated to peg for scavenging organophosphates in circulation
US7067494B2 (en) * 2001-06-22 2006-06-27 The University Of British Columbia Antimitotic eleuthesides
US7229810B2 (en) * 2001-06-28 2007-06-12 Mountain View Pharmaceuticals, Inc. Polymer conjugates of proteinases
US20040077835A1 (en) * 2001-07-12 2004-04-22 Robin Offord Chemokine receptor modulators, production and use
ATE376020T1 (en) 2001-08-22 2007-11-15 Bioartificial Gel Technologies Inc METHOD FOR PRODUCING ACTIVATED POLYETHYLENE GLYCOLS
EP2236161A1 (en) 2001-10-18 2010-10-06 Nektar Therapeutics Polymer conjugates of opioid antagonists
AU2002351388A1 (en) * 2001-12-14 2003-06-30 The University Of Wyoming Methods and compositions for controlled release of drugs
GB0202906D0 (en) 2002-02-07 2002-03-27 Univ London Prevention of myocardial damage
US20030229333A1 (en) * 2002-02-22 2003-12-11 Control Delivery Systems, Inc. Methods for treating otic disorders
EP1364663A1 (en) * 2002-05-21 2003-11-26 Commonwealth Scientific And Industrial Research Organisation Ocular devices with functionalized surface with adhesive properties
EP2298278B1 (en) 2002-06-07 2015-11-11 Dyax Corp. Prevention and reduction of blood loss and inflammatory response
US7153829B2 (en) 2002-06-07 2006-12-26 Dyax Corp. Kallikrein-inhibitor therapies
US7569214B2 (en) 2002-09-09 2009-08-04 Nektar Therapeutics Al, Corporation Method for preparing water-soluble polymer derivatives bearing a terminal carboxylic acid
WO2004035537A2 (en) 2002-10-16 2004-04-29 Euro-Celtique S.A. Antibodies that bind cell-associated ca 125/o772p and methods of use thereof
WO2004061094A1 (en) 2002-12-30 2004-07-22 Gryphon Therapeutics, Inc. Water-soluble thioester and selenoester compounds and methods for making and using the same
US20060014248A1 (en) * 2003-01-06 2006-01-19 Xencor, Inc. TNF super family members with altered immunogenicity
US20050130892A1 (en) * 2003-03-07 2005-06-16 Xencor, Inc. BAFF variants and methods thereof
US7553930B2 (en) * 2003-01-06 2009-06-30 Xencor, Inc. BAFF variants and methods thereof
US20050221443A1 (en) * 2003-01-06 2005-10-06 Xencor, Inc. Tumor necrosis factor super family agonists
US20060104968A1 (en) 2003-03-05 2006-05-18 Halozyme, Inc. Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminogly ycanases
JP4464395B2 (en) * 2003-03-05 2010-05-19 ヘイローザイム インコーポレイテッド Soluble hyaluronidase glycoprotein (sHASEGP), process for its preparation, use and pharmaceutical composition comprising it
US20090123367A1 (en) * 2003-03-05 2009-05-14 Delfmems Soluble Glycosaminoglycanases and Methods of Preparing and Using Soluble Glycosaminoglycanases
US7871607B2 (en) * 2003-03-05 2011-01-18 Halozyme, Inc. Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases
US7610156B2 (en) 2003-03-31 2009-10-27 Xencor, Inc. Methods for rational pegylation of proteins
EP1610825A2 (en) * 2003-03-31 2006-01-04 Xencor, Inc. Methods for rational pegylation of proteins
US7642340B2 (en) 2003-03-31 2010-01-05 Xencor, Inc. PEGylated TNF-α variant proteins
GB0314472D0 (en) * 2003-06-20 2003-07-23 Warwick Effect Polymers Ltd Polymer
EP1665416B1 (en) * 2003-08-01 2014-04-30 Bathium Canada Inc. Cathode material for polymer batteries and method of preparing same
CA3050564A1 (en) * 2003-08-29 2005-03-10 Dyax Corp. Poly-pegylated protease inhibitors
US8668926B1 (en) 2003-09-15 2014-03-11 Shaker A. Mousa Nanoparticle and polymer formulations for thyroid hormone analogs, antagonists, and formulations thereof
US9198887B2 (en) 2003-09-15 2015-12-01 Nanopharmaceuticals Llc Thyroid hormone analogs and methods of use
US7785632B2 (en) 2003-09-15 2010-08-31 Ordway Research Institute, Inc. Thyroid hormone analogs and methods of use
AU2004279895A1 (en) 2003-10-10 2005-04-21 Xencor, Inc Protein based TNF-alpha variants for the treatment of TNF-alpha related disorders
US7786213B2 (en) * 2003-10-15 2010-08-31 The Regents Of The University Of California Biomacromolecule polymer conjugates
US9695251B2 (en) 2003-10-31 2017-07-04 The Regents Of The University Of California Activatable cell penetrating peptides with quenched fluorophores
US7985401B2 (en) * 2003-10-31 2011-07-26 The Regents Of The University Of California Peptides whose uptake by cells is controllable
US7431915B2 (en) * 2003-10-31 2008-10-07 The Regents Of The University Of California Peptides whose uptake by cells is controllable
CN1925875A (en) * 2003-12-16 2007-03-07 尼克塔治疗亚拉巴马公司 Chemically modified small molecules
US20060182692A1 (en) 2003-12-16 2006-08-17 Fishburn C S Chemically modified small molecules
GB2429207A (en) 2004-02-02 2007-02-21 Ambrx Inc Modified human interferon polypeptides and their uses
AU2005319099B2 (en) 2004-02-02 2010-09-16 Ambrx, Inc. Modified human growth hormone
US7351787B2 (en) * 2004-03-05 2008-04-01 Bioartificial Gel Technologies, Inc. Process for the preparation of activated polyethylene glycols
WO2005091944A2 (en) * 2004-03-17 2005-10-06 Eli Lilly And Company Glycol linked fgf-21 compounds
JP4991037B2 (en) * 2004-04-01 2012-08-01 カーディオム ファーマ コーポレイション Drug conjugates comprising ion channel modulating compounds
WO2005115477A2 (en) 2004-04-13 2005-12-08 Quintessence Biosciences, Inc. Non-natural ribonuclease conjugates as cytotoxic agents
WO2005115444A2 (en) * 2004-04-14 2005-12-08 Avirid, Inc. Compositions with modified nucleases targeted to viral nucleic acids and methods of use for prevention and treatment of viral diseases
KR101699142B1 (en) * 2004-06-18 2017-01-23 암브룩스, 인코포레이티드 Novel antigen-binding polypeptides and their uses
WO2006019904A1 (en) 2004-07-14 2006-02-23 University Of Utha Research Foundation Netrin-related compositions and uses
EP1771573A4 (en) * 2004-07-21 2009-02-18 Ambrx Inc Biosynthetic polypeptides utilizing non-naturally encoded amino acids
US7235530B2 (en) 2004-09-27 2007-06-26 Dyax Corporation Kallikrein inhibitors and anti-thrombolytic agents and uses thereof
EP1836298B1 (en) * 2004-12-22 2012-01-18 Ambrx, Inc. COMPOSITIONS OF AMINOACYL-tRNA SYNTHETASE AND USES THEREOF
US8080391B2 (en) 2004-12-22 2011-12-20 Ambrx, Inc. Process of producing non-naturally encoded amino acid containing high conjugated to a water soluble polymer
US7816320B2 (en) 2004-12-22 2010-10-19 Ambrx, Inc. Formulations of human growth hormone comprising a non-naturally encoded amino acid at position 35
DK1835938T3 (en) 2004-12-27 2013-11-04 Baxter Int Polymer-von Willebrand factor conjugates
JP2008530305A (en) * 2005-02-09 2008-08-07 タイコ ヘルスケア グループ エルピー Synthetic sealant
JP2008533114A (en) * 2005-03-18 2008-08-21 ユーシーエル ビジネス パブリック リミテッド カンパニー Mechano growth factor peptides and uses thereof
EP1861125A2 (en) * 2005-03-23 2007-12-05 Nektar Therapeutics Al, Corporation Conjugates of an hgh moiety and peg derivatives
CA2602654A1 (en) 2005-04-05 2006-10-12 Istituto Di Ricerche Di Biologia Molecolare P Angeletti Spa Method for shielding functional sites or epitopes on proteins
US8273339B2 (en) 2005-04-12 2012-09-25 Nektar Therapeutics Polymer-based compositions and conjugates of antimicrobial agents
PT1881850E (en) 2005-05-13 2010-11-26 Lilly Co Eli Glp-1 pegylated compounds
US20060263328A1 (en) * 2005-05-19 2006-11-23 Sang Van Hydrophilic polymers with pendant functional groups and method thereof
AU2006248166B2 (en) * 2005-05-20 2012-11-01 Agency For Science, Technology And Research Aldehyde conjugated flavonoid preparations
US7858080B2 (en) 2005-05-20 2010-12-28 Agency For Science, Technology And Research Aldehyde conjugated flavonoid preparations
ATE529442T1 (en) * 2005-06-03 2011-11-15 Ambrx Inc IMPROVED HUMAN INTERFERON MOLECULES AND THEIR USES
CA2613737C (en) * 2005-06-29 2017-05-23 Yeda Research And Development Co. Ltd. Recombinant interferon .alpha.2 (ifn.alpha.2) mutants
WO2007011802A1 (en) 2005-07-18 2007-01-25 Nektar Therapeutics Al, Corporation Method for preparing branched functionalized polymers using branched polyol cores
US8367096B2 (en) 2005-07-19 2013-02-05 Boston Scientific Scimed, Inc. Polymers having covalently bound therapeutic agents
AU2005335491B2 (en) 2005-08-18 2010-11-25 Ambrx, Inc. Compositions of tRNA and uses thereof
US9498536B2 (en) 2005-09-15 2016-11-22 Nanopharmaceuticals Llc Method and composition of thyroid hormone analogues and nanoformulations thereof for treating anti-inflammatory disorders
US10130686B2 (en) 2005-09-15 2018-11-20 Nanopharmaceuticals Llc Method and composition of thyroid hormone analogues and nanoformulations thereof for treating inflammatory disorders
WO2007035612A2 (en) * 2005-09-16 2007-03-29 Ordway Research Institute, Inc. Polyphenol conjugates as rgd-binding compounds and methods of use
PT2339014E (en) * 2005-11-16 2015-10-13 Ambrx Inc Methods and compositions comprising non-natural amino acids
EP1968644B1 (en) 2005-12-16 2012-06-27 Nektar Therapeutics Polymer conjugates of glp-1
WO2007082061A2 (en) 2006-01-11 2007-07-19 Hyperbranch Medical Technology, Inc. Crosslinked gels comprising polyalkyleneimines, and their uses as medical devices
US9603941B2 (en) * 2006-01-24 2017-03-28 Minghui Chai Method of preparing dendritic drugs
KR101513732B1 (en) * 2006-02-21 2015-04-21 넥타르 테라퓨틱스 Segmented degradable polymers and conjugates made therefrom
ITMI20060612A1 (en) * 2006-03-30 2007-09-30 Keryos Spa NEW ACTIVADED POLY-ETHYLENE GLYCOLS-AND RELATED POLYMERS AND THEIR APPLICATIONS
US7645860B2 (en) 2006-03-31 2010-01-12 Baxter Healthcare S.A. Factor VIII polymer conjugates
US7683158B2 (en) 2006-03-31 2010-03-23 Baxter International Inc. Pegylated factor VIII
US8288339B2 (en) 2006-04-20 2012-10-16 Amgen Inc. GLP-1 compounds
PL2044111T3 (en) 2006-06-21 2015-02-27 Musc Found For Res Dev Targeting complement factor h for treatment of diseases
JP2009541333A (en) 2006-06-23 2009-11-26 クインテセンス バイオサイエンシーズ インコーポレーティッド Modified ribonuclease
EP2049151A4 (en) * 2006-07-17 2010-03-24 Quintessence Biosciences Inc Methods and compositions for the treatment of cancer
US7947758B2 (en) * 2006-08-09 2011-05-24 Ethicon, Inc. Moisture activated latent curing adhesive or sealant
US8129445B2 (en) * 2006-08-09 2012-03-06 Ethicon, Inc. Moisture activated latent curing adhesive or sealant
WO2008030614A2 (en) 2006-09-08 2008-03-13 Ambrx, Inc. Suppressor trna transcription in vertebrate cells
DK2615108T3 (en) * 2006-09-08 2017-01-30 Ambrx Inc Modified human plasma polypeptide or fc scaffolds and their applications
WO2008030613A2 (en) * 2006-09-08 2008-03-13 Ambrx, Inc. Hybrid suppressor trna for vertebrate cells
US7985783B2 (en) 2006-09-21 2011-07-26 The Regents Of The University Of California Aldehyde tags, uses thereof in site-specific protein modification
ES2535005T3 (en) * 2006-12-22 2015-05-04 Nanopharmaceuticals Llc Formulations of nanoparticles and polymers for analogs, antagonists and formulations of thyroid hormone, and uses thereof
LT2842967T (en) 2007-01-18 2017-02-27 Eli Lilly And Company Pegylated amyloid beta fab
JPWO2008105142A1 (en) * 2007-02-28 2010-06-03 日本電気株式会社 Matrix solution
US20090227689A1 (en) * 2007-03-05 2009-09-10 Bennett Steven L Low-Swelling Biocompatible Hydrogels
US20090227981A1 (en) * 2007-03-05 2009-09-10 Bennett Steven L Low-Swelling Biocompatible Hydrogels
KR20140012199A (en) 2007-03-30 2014-01-29 암브룩스, 인코포레이티드 Modified fgf-21 polypeptides and their uses
CN103965347B (en) 2007-05-02 2017-07-18 Ambrx公司 Modified interferon beta polypeptides and its purposes
US20080287633A1 (en) * 2007-05-18 2008-11-20 Drumheller Paul D Hydrogel Materials
US20080300790A1 (en) * 2007-05-29 2008-12-04 James Kirunda Kakaire Environmental data delivery - edd
CN101754951B (en) * 2007-06-26 2013-06-12 巴克斯特国际公司 Method for preparing FMOC-based hydrolysable linkers
US9125807B2 (en) 2007-07-09 2015-09-08 Incept Llc Adhesive hydrogels for ophthalmic drug delivery
US8067028B2 (en) * 2007-08-13 2011-11-29 Confluent Surgical Inc. Drug delivery device
CA2696208A1 (en) * 2007-08-21 2009-02-26 Genzyme Corporation Treatment with kallikrein inhibitors
US8697062B2 (en) * 2007-10-08 2014-04-15 Quintessence Biosciences, Inc. Compositions and methods for ribonuclease-based therapeutics
AU2008326324B9 (en) 2007-11-20 2012-11-15 Ambrx, Inc. Modified insulin polypeptides and their uses
EP3103880A1 (en) 2008-02-08 2016-12-14 Ambrx, Inc. Modified leptin polypeptides and their uses
TWI395593B (en) 2008-03-06 2013-05-11 Halozyme Inc In vivo temporal control of activatable matrix-degrading enzymes
EP2285402A2 (en) 2008-04-14 2011-02-23 Halozyme, Inc. Modified hyaluronidases and uses in treating hyaluronan-associated diseases and conditions
TWI394580B (en) 2008-04-28 2013-05-01 Halozyme Inc Super fast-acting insulin compositions
UA118536C2 (en) 2008-07-23 2019-02-11 Амбркс, Інк. MODIFIED Bovine granulocyte colony-stimulating factor polypeptide and its application
KR101660059B1 (en) * 2008-08-22 2016-09-26 박스알타 인코퍼레이티드 Polymeric benzyl carbonate-derivatives
EP2340050A2 (en) * 2008-09-19 2011-07-06 Nektar Therapeutics Polymer conjugates of aod-like peptides
MX2011003117A (en) 2008-09-19 2011-04-21 Nektar Therapeutics Polymer conjugates of therapeutic peptides.
US20110171165A1 (en) * 2008-09-19 2011-07-14 Nektar Therapeutics Polymer conjugates of opioid growth factor peptides
US20110171161A1 (en) * 2008-09-19 2011-07-14 Nektar Therapeutics Polymer conjugates of protegrin peptides
US20110206633A1 (en) * 2008-09-19 2011-08-25 Nektar Therapectics Polymer conjugates of cd-np peptides
WO2010033227A1 (en) * 2008-09-19 2010-03-25 Nektar Therapeutics Polymer conjugates of thymosin alpha 1 peptides
EP2334333A1 (en) * 2008-09-19 2011-06-22 Nektar Therapeutics Polymer conjugates of v681-like peptides
EP2344200A2 (en) * 2008-09-19 2011-07-20 Nektar Therapeutics Modified therapeutics peptides, methods of their preparation and use
US20110171166A1 (en) * 2008-09-19 2011-07-14 Nektar Therapeutics Polymer conjugates of osteocalcin peptides
US20110165112A1 (en) * 2008-09-19 2011-07-07 Nektar Therapeutics Polymer conjugates of c-peptides
US20110171164A1 (en) * 2008-09-19 2011-07-14 Nektar Therapeutics Polymer conjugates of glp-2-like peptides
EP2340047A1 (en) * 2008-09-19 2011-07-06 Nektar Therapeutics Polymer conjugates of kiss1 peptides
WO2010033222A2 (en) * 2008-09-19 2010-03-25 Netkar Therapeutics Polymer conjugates of ziconotide peptides
WO2010034015A2 (en) 2008-09-22 2010-03-25 The Regents Of The University Of Colorado, A Body Corporate Modulating the alternative complement pathway
EP3216800A1 (en) 2008-09-26 2017-09-13 Ambrx, Inc. Modified animal erythropoietin polypeptides and their uses
MX348657B (en) 2008-09-26 2017-06-21 Ambrx Inc Non-natural amino acid replication-dependent microorganisms and vaccines.
EP2334695B1 (en) 2008-10-01 2015-12-23 Quintessence Biosciences, Inc. Therapeutic ribonucleases
US9274122B2 (en) * 2008-10-21 2016-03-01 Baxalta Incorporated Methods for determining active ingredients in pro-drug PEG protein conjugates with releasable PEG reagents (in vitro de-pegylation)
DK3037529T3 (en) 2008-12-09 2019-05-20 Halozyme Inc EXTENDED SOLUBLE PH20 POLYPEPTIDES AND USE THEREOF
WO2010075332A1 (en) * 2008-12-23 2010-07-01 Charitable Leadership Foundation Small molecule ligands of the integrin rgd recognition site and methods of use
AU2010203712A1 (en) * 2009-01-06 2010-07-15 Dyax Corp. Treatment of mucositis with kallikrein inhibitors
JP5890182B2 (en) 2009-02-12 2016-03-22 インセプト エルエルシー Drug delivery with hydrogel plugs
US9180107B2 (en) * 2009-03-31 2015-11-10 Nanopharmaceuticals Llc Combination treatment of cancer with cetuximab and tetrac
CN101870728A (en) 2009-04-23 2010-10-27 派格生物医药(苏州)有限公司 Novel Exendin variant and conjugate thereof
WO2010148007A2 (en) 2009-06-17 2010-12-23 Ordway Research Institute, Inc. Nanoparticle and polymer formulations for thyroid hormone, analogs, antagonists, and formulations and uses thereof
EP2453906A4 (en) 2009-07-02 2014-01-15 Musc Found For Res Dev Methods of stimulating liver regeneration
EP2454271A4 (en) 2009-07-15 2015-08-12 Univ California Peptides whose uptake in cells is controllable
JP5734985B2 (en) 2009-09-17 2015-06-17 バクスター・ヘルスケヤー・ソシエテ・アノニムBaxter Healthcare SA Stable co-formulations of hyaluronidase and immunoglobulin and methods for their use
CN102666845B (en) 2009-10-16 2015-06-10 不列颠哥伦比亚大学 Inhibitors of phosphatase and tensin homolog (PTEN) compositions, uses and methods
US8138236B2 (en) * 2009-10-29 2012-03-20 Ethicon, Inc. Solvent-free moisture activated latent curing surgical adhesive or sealant
CN102958535A (en) 2009-11-05 2013-03-06 亚力史剑桥公司 Treatment of paroxysmal nocturnal hemoglobinuria, hemolytic anemias and disease states involving intravascular and extravascular hemolysis
CA3042067C (en) 2009-12-15 2022-10-18 Incept, Llc Implants and biodegradable fiducial markers
CN102753573A (en) 2009-12-21 2012-10-24 Ambrx公司 Modified bovine somatotropin polypeptides and their uses
NZ600363A (en) 2009-12-21 2014-07-25 Ambrx Inc Modified porcine somatotropin polypeptides and their uses
AR079345A1 (en) 2009-12-22 2012-01-18 Lilly Co Eli OXINTOMODULINE PEPTIDAL ANALOG
AR079344A1 (en) 2009-12-22 2012-01-18 Lilly Co Eli PEPTIDAL ANALOG OF OXINTOMODULIN, PHARMACEUTICAL COMPOSITION THAT UNDERSTANDS AND USES TO PREPARE A USEFUL MEDICINAL PRODUCT TO TREAT NON-INSULINED INDEPENDENT DIABETES AND / OR OBESITY
LT2521568T (en) 2010-01-06 2018-12-10 Dyax Corp. Plasma kallikrein binding proteins
SA111320200B1 (en) 2010-02-17 2014-02-16 ديبيوفارم اس ايه Bicyclic Compounds and their Uses as Dual C-SRC / JAK Inhibitors
BR112012026372A2 (en) * 2010-04-16 2016-08-02 Apatech Ltd biocompatible material, method for making a material, and resorbable polymer matrix
WO2011141891A1 (en) 2010-05-12 2011-11-17 Debio Recherche Pharmaceutique S.A. Use of cycloundecadepsipeptide compounds
CN103038252A (en) 2010-05-14 2013-04-10 科罗拉多大学董事会,法人团体 Improved complement receptor 2 (CR2) targeting groups
AU2011270959A1 (en) 2010-06-22 2013-01-10 Musc Foundation For Research Development Antibodies to the C3d fragment of complement component 3
US9878046B2 (en) 2010-07-20 2018-01-30 Halozyme, Inc. Adverse side-effects associated with administration of an anti-hyaluronan agent and methods for ameliorating or preventing the side-effects
CA2807552A1 (en) 2010-08-06 2012-02-09 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9567386B2 (en) 2010-08-17 2017-02-14 Ambrx, Inc. Therapeutic uses of modified relaxin polypeptides
BR112013003522B1 (en) 2010-08-17 2021-05-25 Ambrx, Inc. modified relaxin polypeptides comprising a non-naturally encoded amino acid, their method of preparation and their use, as well as nucleic acid and host cell
US8961501B2 (en) 2010-09-17 2015-02-24 Incept, Llc Method for applying flowable hydrogels to a cornea
TWI480288B (en) 2010-09-23 2015-04-11 Lilly Co Eli Formulations for bovine granulocyte colony stimulating factor and variants thereof
CN104531812A (en) 2010-10-01 2015-04-22 现代治疗公司 Engineered nucleic acids and methods of use thereof
US8802240B2 (en) 2011-01-06 2014-08-12 Nanopharmaceuticals Llc Uses of formulations of thyroid hormone analogs and nanoparticulate forms thereof to increase chemosensitivity and radiosensitivity in tumor or cancer cells
BR112013017080A8 (en) 2011-01-06 2023-05-09 Dyax Corp ANTIBODY OR FUNCTIONAL FRAGMENT THEREOF WHICH BINDS THE ACTIVE FORM OF HUMAN PLASMA KALLICREIN, PHARMACEUTICAL COMPOSITION AND METHOD OF DETECTION OF PLASMA KALLICREIN IN A PATIENT
US8440309B2 (en) 2011-01-31 2013-05-14 Confluent Surgical, Inc. Crosslinked polymers with the crosslinker as therapeutic for sustained release
WO2012109387A1 (en) 2011-02-08 2012-08-16 Halozyme, Inc. Composition and lipid formulation of a hyaluronan-degrading enzyme and the use thereof for treatment of benign prostatic hyperplasia
WO2012135805A2 (en) 2011-03-31 2012-10-04 modeRNA Therapeutics Delivery and formulation of engineered nucleic acids
US9993529B2 (en) 2011-06-17 2018-06-12 Halozyme, Inc. Stable formulations of a hyaluronan-degrading enzyme
CN103889443A (en) 2011-06-17 2014-06-25 哈洛齐梅公司 Continuous subcutaneous insulin infusion methods with a hyaluronan degrading enzyme
PL2717917T3 (en) 2011-07-05 2016-12-30 P97-antibody conjugates
RU2014103185A (en) 2011-07-18 2015-08-27 Артс Байолоджикс А/С BIOLOGICALLY ACTIVE COMPOUND BASED ON LUTEINIZING HORMONE (LH) WITH PROLONGED ACTION
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US20130071394A1 (en) 2011-09-16 2013-03-21 John K. Troyer Compositions and combinations of organophosphorus bioscavengers and hyaluronan-degrading enzymes, and methods of use
US10226417B2 (en) 2011-09-16 2019-03-12 Peter Jarrett Drug delivery systems and applications
RU2707251C2 (en) 2011-10-03 2019-11-25 Модерна Терапьютикс, Инк. Modified nucleosides, nucleotides and nucleic acids and use thereof
AU2012328880B2 (en) 2011-10-24 2017-02-23 Halozyme, Inc. Companion diagnostic for anti-hyaluronan agent therapy and methods of use thereof
KR20190090048A (en) 2011-12-05 2019-07-31 인셉트, 엘엘씨 Medical organogel processes and compositions
KR20140102759A (en) 2011-12-16 2014-08-22 모더나 세라퓨틱스, 인코포레이티드 Modified nucleoside, nucleotide, and nucleic acid compositions
EP2606884A1 (en) 2011-12-21 2013-06-26 Ecole Polytechnique Fédérale de Lausanne (EPFL) Inhibitors of notch signaling pathway and use thereof in treatment of cancers
IL298330A (en) 2011-12-30 2023-01-01 Halozyme Inc Ph20 polypeptide variants, formulations and uses thereof
WO2013149161A1 (en) 2012-03-30 2013-10-03 Deangelis Paul L High molecular weight heparosan polymers and methods of production and use thereof
EP2833892A4 (en) 2012-04-02 2016-07-20 Moderna Therapeutics Inc Modified polynucleotides for the production of oncology-related proteins and peptides
US9878056B2 (en) 2012-04-02 2018-01-30 Modernatx, Inc. Modified polynucleotides for the production of cosmetic proteins and peptides
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
EA031986B1 (en) 2012-04-04 2019-03-29 Галозим, Инк. Method and combination for treating solid tumor cancer and kit comprising the combination
EP2846822A2 (en) 2012-05-11 2015-03-18 Prorec Bio AB Method for diagnosis and treatment of prolactin associated disorders
AU2013265266A1 (en) 2012-05-23 2015-01-15 Stemergie Biotechnology Sa Inhibitors of the activity of complex (III) of the mitochondrial electron transport chain and use thereof
EP3505534A1 (en) 2012-06-08 2019-07-03 Sutro Biopharma, Inc. Antibodies comprising sitespecific nonnatural amino acid residues, methods of their preparation and methods of their use
ES2611788T3 (en) 2012-06-26 2017-05-10 Sutro Biopharma, Inc. Modified Fc proteins comprising site-specific non-natural amino acid residues, conjugates thereof, methods for their preparation and methods for use
JP6433424B2 (en) 2012-07-31 2018-12-05 バイオアシス テクノロジーズ インコーポレイテッド Dephosphorylated lysosomal storage disease protein and method of use thereof
US10413620B2 (en) 2012-08-17 2019-09-17 The Regents Of The University Of Colorado, A Body Corporate Light-emitting versions of the monoclonal antibody to C3D (MAB 3D29) for imaging
AU2013302441B2 (en) 2012-08-17 2018-05-10 The Regents Of The University Of Colorado, A Body Corporate Compositions and methods for detecting complement activation
BR112015004022B1 (en) 2012-08-31 2023-04-25 Sutro Biopharma, Inc MODIFIED AMINO ACIDS COMPRISING AN AZID GROUP
US9278124B2 (en) 2012-10-16 2016-03-08 Halozyme, Inc. Hypoxia and hyaluronan and markers thereof for diagnosis and monitoring of diseases and conditions and related methods
CA2890906A1 (en) 2012-11-16 2014-05-22 The Regents Of The University Of California Pictet-spengler ligation for protein chemical modification
US9310374B2 (en) 2012-11-16 2016-04-12 Redwood Bioscience, Inc. Hydrazinyl-indole compounds and methods for producing a conjugate
JP6144355B2 (en) 2012-11-26 2017-06-07 モデルナティエックス インコーポレイテッドModernaTX,Inc. Chemically modified mRNA
WO2014120837A2 (en) 2013-01-29 2014-08-07 The Regents Of The University Of California Pretargeted activatable cell penetrating peptide with intracellulary releaseable prodrug
EA031930B1 (en) * 2013-01-30 2019-03-29 Авелас Байосайенсиз, Инк. Selective delivery molecule for visualizing a cancerous tissue
BR112015022416A2 (en) 2013-03-13 2017-10-24 Bioasis Technologies Inc p97 fragments and their uses
WO2014152211A1 (en) 2013-03-14 2014-09-25 Moderna Therapeutics, Inc. Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
TW201534726A (en) 2013-07-03 2015-09-16 Halozyme Inc Thermally stable PH20 hyaluronidase variants and uses thereof
US9764039B2 (en) 2013-07-10 2017-09-19 Sutro Biopharma, Inc. Antibodies comprising multiple site-specific non-natural amino acid residues, methods of their preparation and methods of their use
JP2016531910A (en) 2013-08-16 2016-10-13 アレクシオン ファーマシューティカルズ, インコーポレイテッド Treatment of transplant rejection by administration of complement inhibitors to pre-transplant organs
WO2015031673A2 (en) 2013-08-28 2015-03-05 Bioasis Technologies Inc. Cns-targeted conjugates having modified fc regions and methods of use thereof
US20160194368A1 (en) 2013-09-03 2016-07-07 Moderna Therapeutics, Inc. Circular polynucleotides
AU2014315287A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Chimeric polynucleotides
WO2015048744A2 (en) 2013-09-30 2015-04-02 Moderna Therapeutics, Inc. Polynucleotides encoding immune modulating polypeptides
US10323076B2 (en) 2013-10-03 2019-06-18 Modernatx, Inc. Polynucleotides encoding low density lipoprotein receptor
WO2015054658A1 (en) 2013-10-11 2015-04-16 Sutro Biopharma, Inc. Modified amino acids comprising tetrazine functional groups, methods of preparation, and methods of their use
WO2015081282A1 (en) 2013-11-27 2015-06-04 Redwood Bioscience, Inc. Hydrazinyl-pyrrolo compounds and methods for producing a conjugate
WO2015081891A1 (en) 2013-12-06 2015-06-11 Baikang (Suzhou) Co., Ltd Bioreversable promoieties for nitrogen-containing and hydroxyl-containing drugs
US10428158B2 (en) 2014-03-27 2019-10-01 Dyax Corp. Compositions and methods for treatment of diabetic macular edema
WO2015175774A1 (en) 2014-05-14 2015-11-19 Trustees Of Dartmouth College Deimmunized lysostaphin and methods of use
CA2955250A1 (en) 2014-07-16 2016-01-21 Moderna Therapeutics, Inc. Chimeric polynucleotides
US20170210788A1 (en) 2014-07-23 2017-07-27 Modernatx, Inc. Modified polynucleotides for the production of intrabodies
HUE043847T2 (en) 2014-08-28 2019-09-30 Halozyme Inc Combination therapy with a hyaluronan-degrading enzyme and an immune checkpoint inhibitor
US10385380B2 (en) 2014-10-02 2019-08-20 The Regents Of The University Of California Personalized protease assay to measure protease activity in neoplasms
EA201700181A1 (en) 2014-10-14 2017-09-29 Галозим, Инк. COMPOSITIONS OF ADENOSINDEMINASE-2 (ADA-2), THEIR OPTIONS AND METHODS OF USE
KR20240024362A (en) 2014-10-24 2024-02-23 브리스톨-마이어스 스큅 컴퍼니 Modified fgf-21 polypeptides and uses thereof
EP4279064A3 (en) 2015-05-12 2024-02-28 Incept, LLC Drug delivery from hydrogels
US10596259B2 (en) 2015-05-20 2020-03-24 The Regents Of The University Of California Tumor radiosensitization with monomethyl auristatin E (MMAE) and derivatives thereof
JP6921833B2 (en) 2015-10-22 2021-08-18 モデルナティーエックス, インコーポレイテッド Human cytomegalovirus vaccine
BR112018008090A2 (en) 2015-10-22 2018-11-13 Modernatx Inc herpes simplex virus vaccine.
JP7384512B2 (en) 2015-10-22 2023-11-21 モデルナティエックス インコーポレイテッド Broad-spectrum influenza virus vaccine
TW201729838A (en) 2015-10-22 2017-09-01 現代公司 Nucleic acid vaccines for varicella zoster virus (VZV)
EP3365008A4 (en) 2015-10-22 2019-08-07 ModernaTX, Inc. Respiratory syncytial virus vaccine
MA47016A (en) 2015-10-22 2018-08-29 Modernatx Inc RESPIRATORY VIRUS VACCINES
BR112018011622A2 (en) 2015-12-11 2018-11-27 Dyax Corp method to treat hereditary angioedema attack (hae) or reduce hae attack rate
ES2919552T3 (en) 2015-12-23 2022-07-27 Modernatx Inc Methods of using ox40 ligand-encoding polynucleotides
EP3400023A1 (en) 2016-01-10 2018-11-14 ModernaTX, Inc. Therapeutic mrnas encoding anti ctla-4 antibodies
WO2017214299A1 (en) 2016-06-07 2017-12-14 Nanopharmaceuticals, Llc NON-CLEAVABLE POLYMER CONJUGATED WITH αvβ3 INTEGRIN THYROID ANTAGONISTS
US10988529B2 (en) 2016-08-09 2021-04-27 Eli Lilly And Company Combination therapy
CN110637027A (en) 2017-02-08 2019-12-31 百时美施贵宝公司 Modified relaxin polypeptides comprising pharmacokinetic enhancers and uses thereof
CA3063723A1 (en) 2017-05-18 2018-11-22 Modernatx, Inc. Polynucleotides encoding tethered interleukin-12 (il12) polypeptides and uses thereof
US10781435B2 (en) 2017-06-22 2020-09-22 Catalyst Biosciences, Inc. Modified membrane type serine protease 1 (MTSP-1) polypeptides and methods of use
WO2019063958A1 (en) 2017-09-27 2019-04-04 The University Of York Bioconjugation of polypeptides
US11492493B2 (en) 2017-12-26 2022-11-08 Becton, Dickinson And Company Deep ultraviolet-excitable water-solvated polymeric dyes
CA3095644A1 (en) * 2018-03-29 2019-10-03 Nof Corporation Degradable polyethylene glycol conjugate
EP3775052B1 (en) 2018-03-30 2024-06-05 Becton, Dickinson and Company Water-soluble polymeric dyes having pendant chromophores
US10328043B1 (en) 2018-04-11 2019-06-25 Nanopharmaceuticals, Llc. Composition and method for dual targeting in treatment of neuroendocrine tumors
US11351137B2 (en) 2018-04-11 2022-06-07 Nanopharmaceuticals Llc Composition and method for dual targeting in treatment of neuroendocrine tumors
WO2019222435A1 (en) 2018-05-16 2019-11-21 Halozyme, Inc. Methods of selecting subjects for combination cancer therapy with a polymer-conjugated soluble ph20
CA3107332A1 (en) 2018-07-22 2020-01-30 Bioasis Technologies Inc. Treatment of lymphatic metastases
US20220056093A1 (en) 2018-09-11 2022-02-24 Ambrx, Inc. Interleukin-2 polypeptide conjugates and their uses
CN110964116A (en) 2018-09-26 2020-04-07 北京辅仁瑞辉生物医药研究院有限公司 GLP1-Fc fusion proteins and conjugates thereof
EP3867265A1 (en) 2018-10-19 2021-08-25 Ambrx, Inc. Interleukin-10 polypeptide conjugates, dimers thereof, and their uses
US20200262887A1 (en) 2018-11-30 2020-08-20 Opko Ireland Global Holdings, Ltd. Oxyntomodulin peptide analog formulations
EP3902913A1 (en) 2018-12-28 2021-11-03 Catalyst Biosciences, Inc. Modified urokinase-type plasminogen activator polypeptides and methods of use
US11613744B2 (en) 2018-12-28 2023-03-28 Vertex Pharmaceuticals Incorporated Modified urokinase-type plasminogen activator polypeptides and methods of use
BR112021015832A2 (en) 2019-02-12 2022-01-18 Ambrx Inc Compositions containing antibody-tlr agonist conjugates, methods and uses thereof
US20220169642A1 (en) 2019-04-10 2022-06-02 Cellestia Biotech Ag Compounds for the treatment of oncovirus induced cancer and methods of use thereof
CN112175088B (en) 2019-07-02 2023-03-28 江苏晟斯生物制药有限公司 FIX fusion proteins, conjugates and uses thereof
US11793881B2 (en) * 2019-11-28 2023-10-24 Chongqing Upgra Biotechnology Co., Ltd. Polyethylene glycol conjugate medicament, preparation method therefor and use thereof
CA3166509A1 (en) 2020-01-14 2021-07-22 Synthekine, Inc. Biased il2 muteins methods and compositions
KR20220151202A (en) 2020-03-11 2022-11-14 암브룩스, 인코포레이티드 Interleukin-2 polypeptide conjugates and methods of use thereof
US10961204B1 (en) 2020-04-29 2021-03-30 Nanopharmaceuticals Llc Composition of scalable thyrointegrin antagonists with improved blood brain barrier penetration and retention into brain tumors
US20210355468A1 (en) 2020-05-18 2021-11-18 Bioasis Technologies, Inc. Compositions and methods for treating lewy body dementia
US20210393787A1 (en) 2020-06-17 2021-12-23 Bioasis Technologies, Inc. Compositions and methods for treating frontotemporal dementia
CA3128035A1 (en) 2020-08-13 2022-02-13 Bioasis Technologies, Inc. Combination therapies for delivery across the blood brain barrier
JP2023538071A (en) 2020-08-20 2023-09-06 アンブルックス,インコーポレイテッド Antibody-TLR agonist conjugates, methods and uses thereof
EP4255409A1 (en) 2020-12-07 2023-10-11 Cellestia Biotech AG Pharmaceutical combinations for treating cancer
EP4008324A1 (en) 2020-12-07 2022-06-08 Cellestia Biotech AG Combinations comprising an inhibitor of an anti-apoptotic protein, such as bcl-2, bcl-xl, bclw or mcl-1, and a notch signaling pathway inhibitor for treating cancer
CA3213805A1 (en) 2021-04-03 2022-10-06 Feng Tian Anti-her2 antibody-drug conjugates and uses thereof
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WO2023079132A1 (en) 2021-11-08 2023-05-11 Cellestia Biotech Ag Pharmaceutical combinations for treating cancer
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WO2024007016A2 (en) 2022-07-01 2024-01-04 Beckman Coulter, Inc. Novel fluorescent dyes and polymers from dihydrophenanthrene derivatives
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Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) * 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US4670417A (en) * 1985-06-19 1987-06-02 Ajinomoto Co., Inc. Hemoglobin combined with a poly(alkylene oxide)
US4766106A (en) * 1985-06-26 1988-08-23 Cetus Corporation Solubilization of proteins for pharmaceutical compositions using polymer conjugation
US5476653A (en) * 1992-06-17 1995-12-19 Amgen Inc. Polyoxymethylene-oxyethylene copolymers in conjuction with biomolecules
US5672662A (en) * 1995-07-07 1997-09-30 Shearwater Polymers, Inc. Poly(ethylene glycol) and related polymers monosubstituted with propionic or butanoic acids and functional derivatives thereof for biotechnical applications
US5720950A (en) * 1990-05-14 1998-02-24 University Of Medicine & Dentistry Of New Jersey Polymers containing antifibrotic agents, compositions containing such polymers, and methods of preparation and use
US5730990A (en) * 1994-06-24 1998-03-24 Enzon, Inc. Non-antigenic amine derived polymers and polymer conjugates
US5738846A (en) * 1994-11-10 1998-04-14 Enzon, Inc. Interferon polymer conjugates and process for preparing the same
US5840900A (en) * 1993-10-20 1998-11-24 Enzon, Inc. High molecular weight polymer-based prodrugs
US6214966B1 (en) * 1996-09-26 2001-04-10 Shearwater Corporation Soluble, degradable poly(ethylene glycol) derivatives for controllable release of bound molecules into solution
US20050158273A1 (en) * 1997-09-25 2005-07-21 Harris J. M. Soluble, degradable polyethylene glycol) derivatives for controllable release of bound molecules into solution

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5349001A (en) 1993-01-19 1994-09-20 Enzon, Inc. Cyclic imide thione activated polyalkylene oxides
US5643575A (en) * 1993-10-27 1997-07-01 Enzon, Inc. Non-antigenic branched polymer conjugates
US5932462A (en) * 1995-01-10 1999-08-03 Shearwater Polymers, Inc. Multiarmed, monofunctional, polymer for coupling to molecules and surfaces

Patent Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) * 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US4670417A (en) * 1985-06-19 1987-06-02 Ajinomoto Co., Inc. Hemoglobin combined with a poly(alkylene oxide)
US4766106A (en) * 1985-06-26 1988-08-23 Cetus Corporation Solubilization of proteins for pharmaceutical compositions using polymer conjugation
US5720950A (en) * 1990-05-14 1998-02-24 University Of Medicine & Dentistry Of New Jersey Polymers containing antifibrotic agents, compositions containing such polymers, and methods of preparation and use
US5476653A (en) * 1992-06-17 1995-12-19 Amgen Inc. Polyoxymethylene-oxyethylene copolymers in conjuction with biomolecules
US5840900A (en) * 1993-10-20 1998-11-24 Enzon, Inc. High molecular weight polymer-based prodrugs
US5730990A (en) * 1994-06-24 1998-03-24 Enzon, Inc. Non-antigenic amine derived polymers and polymer conjugates
US5738846A (en) * 1994-11-10 1998-04-14 Enzon, Inc. Interferon polymer conjugates and process for preparing the same
US5672662A (en) * 1995-07-07 1997-09-30 Shearwater Polymers, Inc. Poly(ethylene glycol) and related polymers monosubstituted with propionic or butanoic acids and functional derivatives thereof for biotechnical applications
US6214966B1 (en) * 1996-09-26 2001-04-10 Shearwater Corporation Soluble, degradable poly(ethylene glycol) derivatives for controllable release of bound molecules into solution
US6515100B2 (en) * 1996-09-26 2003-02-04 Shearwater Corporation Soluble, degradable poly (ethylene glycol) derivatives for controllable release of bound molecules into solution
US6864350B2 (en) * 1996-09-26 2005-03-08 Nektar Therapeutics Al, Corporation Soluble, degradable poly (ethylene glycol) derivatives for controllable release of bound molecules into solution
US20050171328A1 (en) * 1996-09-26 2005-08-04 Nektar Therapeutics Al, Corporation Soluble, degradable poly(ethylene glycol) derivatives for conrollable release of bound molecules into solution
US7655747B2 (en) * 1996-09-26 2010-02-02 Nektar Therapeutics Soluble, degradable poly(ethylene glycol) derivatives for controllable release of bound molecules into solution
US20050158273A1 (en) * 1997-09-25 2005-07-21 Harris J. M. Soluble, degradable polyethylene glycol) derivatives for controllable release of bound molecules into solution

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8404222B2 (en) 1996-09-26 2013-03-26 Nektar Therapeutics Soluble, degradable poly(ethylene glycol) derivatives for controllable release of bound molecules into solution

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US20120071627A1 (en) 2012-03-22
US8088365B2 (en) 2012-01-03
US8404222B2 (en) 2013-03-26
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